High prevalence of BRCA1 founder mutations in Greek breast/ovarian families.

We have screened 473 breast/ovarian cancer patients with family history, aiming to define the prevalence and enrich the spectrum of BRCA1/2 pathogenic mutations occurring in the Greek population. An overall mutation prevalence of 32% was observed. Six BRCA1 recurrent/founder mutations dominate the observed spectrum (58.5% of all mutations found). These include three mutations in exon 20 and three large genomic deletions. Of the 44 different deleterious mutations found in both genes, 16 are novel and reported here for the first time. Correlation with available histopathology data showed that 80% of BRCA1 carriers presented a triple-negative breast cancer phenotype while 82% of BRCA2 carriers had oestrogen receptor positive tumours. This study provides a comprehensive view of the frequency, type and distribution of BRCA1/2 mutations in the Greek population as well as an insight of the screening strategy of choice for patients of Greek origin. We conclude that the Greek population has a diverse mutation spectrum influenced by strong founder effects.

Clinical experience of using Oncotype DX as an additional treatment decision tool in early breast cancer - a retrospective analysis from 5 Greek institutions.

AIMS: The objective of this retrospective study was to describe the results from five institutions' experience of using Oncotype DX(®) to identify patients who need chemotherapy despite the presence of primarily favorable characteristics. PATIENTS AND METHODS: Oncotype DX was performed in 106 pre- and postmenopausal patients with estrogen receptor-positive, HER2-negative, early breast cancer with a combination of favorable prognostic factors or favorable prognostic factors with at least one unfavorable characteristic (tumor size >2 cm, tumor grading of II-III, Ki-67 ≥ 10%, presence of lymph node micrometastases) in which it was unclear whether hormonal therapy only or chemotherapy plus hormonal therapy was the optimal adjuvant treatment. RESULTS: Sixty-four (60.4%) women had Recurrence Score (RS) values <18, 29 (27.4%) intermediate RS values of 18-30, and 13 (12.3%) high RS values of ≥31. Tumor size, grading and presence of micrometastases were not associated with the RS. There was a significant association between Recurrence Score and the number of unfavorable characteristics as a categorical but not as a continuous variable. High Recurrence Scores were predictive of high Ki-67 but the converse was not true. Overall, 29 of 106 (27.4%) patients received chemotherapy because of an intermediate or a high Recurrence Score. CONCLUSION: The Recurrence Score helped in treatment decision-making for estrogen receptor-positive, HER2-negative patients with favorable characteristics or an intermediate risk of recurrence due to the presence of at least one unfavorable factor. The results of the 21-gene assay increased the likelihood for patients with intermediate clinical and histopathological risk factors receiving chemotherapy.

Comparative analysis of in vitro osteo/odontogenic differentiation potential of human dental pulp stem cells (DPSCs) and stem cells from the apical papilla (SCAP).

OBJECTIVE: The aim of this study was to compare the in vitro osteo/odontogenic differentiation potential of mesenchymal stem cells (MSCs) derived from the dental pulp (dental pulp stem cells - DPSCs) or the apical papilla (stem cells from the apical papilla - SCAP) of permanent developing teeth. DESIGN: DPSCs and SCAP cultures were established from impacted third molars of young healthy donors at the stage of root development. Cultures were analysed for stem cell markers, including STRO-1, CD146, CD34 and CD45 using flow cytometry. Cells were then induced for osteo/odontogenic differentiation by media containing dexamethasone, KH(2)PO(4) and ß-glycerophosphate. Cultures were analysed for morphology, growth characteristics, mineralization potential (Alizarin Red method) and differentiation markers (dentine sialophosphoprotein-DSPP, bone sialoprotein-BSP, osteocalcin-OCN, alkaline phosphatase-ALP), using immunocytochemistry and reverse transcriptase-polymerase chain reaction. RESULTS: All DPSCs and SCAP cultures were positive for STRO-1, CD146 and CD34, in percentages varying according to cell type and donor, but negative for CD45. Both types of MSCs displayed an active potential for cellular migration, organization and mineralization, producing 3D mineralized structures. These structures progressively expressed differentiation markers, including DSPP, BSP, OCN, ALP, having the characteristics of osteodentin. SCAP, however, showed a significantly higher proliferation rate and mineralization potential, which might be of significance for their use in bone/dental tissue engineering. CONCLUSIONS: This study provides evidence that different types of dental MSCs can be used in tissue engineering/regeneration protocols as an approachable stem cell source for osteo/odontogenic differentiation and biomineralization that could be further applied for stem cell-based clinical therapies.

On the intracellular trafficking of mouse S5 ribosomal protein from cytoplasm to nucleoli.

The non-ribosomal functions of mammalian ribosomal proteins have recently attracted worldwide attention. The mouse ribosomal protein S5 (rpS5) derived from ribosomal material is an assembled non-phosphorylated protein. The free form of rpS5 protein, however, undergoes phosphorylation. In this study, we have (a) investigated the potential role of phosphorylation in rpS5 protein transport into the nucleus and then into nucleoli and (b) determined which of the domains of rpS5 are involved in this intracellular trafficking. In vitro PCR mutagenesis of mouse rpS5 cDNA, complemented by subsequent cloning and expression of rpS5 truncated recombinant forms, produced in fusion with green fluorescent protein, permitted the investigation of rpS5 intracellular trafficking in HeLa cells using confocal microscopy complemented by Western blot analysis. Our results indicate the following: (a) rpS5 protein enters the nucleus via the region 38-50 aa that forms a random coil as revealed by molecular dynamic simulation. (b) Immunoprecipitation of rpS5 with casein kinase II and immobilized metal affinity chromatography analysis complemented by in vitro kinase assay revealed that phosphorylation of rpS5 seems to be indispensable for its transport from nucleus to nucleoli; upon entering the nucleus, Thr-133 phosphorylation triggers Ser-24 phosphorylation by casein kinase II, thus promoting entrance of rpS5 into the nucleoli. Another important role of rpS5 N-terminal region is proposed to be the regulation of protein's cellular level. The repetitively co-appearance of a satellite C-terminal band below the entire rpS5 at the late stationary phase, and not at the early logarithmic phase, of cell growth suggests a specific degradation balancing probably the unassembled ribosomal protein molecules with those that are efficiently assembled to ribosomal subunits. Overall, these data provide new insights on the structural and functional domains within the rpS5 molecule that contribute to its cellular functions.

Genotoxic and cytotoxic effects of different types of dental cement on normal cultured human lymphocytes.

In this study we have investigated the genotoxic and cytotoxic effects of eluates derived from different types of commercially available dental cements, including glass ionomer cements (GICs) (Ketac Cem/3M ESPE and GC Fuji I/GC Corp), resin-modified glass ionomer cements (RM-GICs) (RelyX Luting/3M ESPE and Vitrebond/3M ESPE) and dual-cure resin cements (RCs) (Variolink II/ Ivoclar-Vivadent and Panavia F 2.0/Kuraray) on normal cultured human lymphocytes. Lymphocyte primary cultures obtained from blood samples of three healthy donors were exposed to serial dilutions of eluates derived from specimens of each material tested. Metaphases were induced with phytohaemagglutinin, collected after 72h treatment by use of colchicine and stained according to the fluorescence plus giemsa (FPG) procedure. Preparations were scored for sister chromatid exchange (SCE) and chromosomal aberrations (CAs), while the proliferation rate index (PRI) was also calculated. Our results show that eluates derived from the RM-GICs and RCs caused severe genotoxic effects by significantly increasing the frequencies of SCEs and CAs in cultures of peripheral blood lymphocytes and by decreasing the relevant PRI values in a dose-dependent manner, whereas the two GICs caused only minor cytogenetic effects. Eluates of the two RM-GICs (Vitrebond and RelyX) were also very cytotoxic, as the first serial dilutions of both materials caused a complete mitotic arrest in lymphocyte cultures. Overall, the degree of genotoxicity and cytotoxicity caused by dental cements decreased as follows: Viterbond>Rely X>Panavia F 2.0>Variolink II>Ketac Cem=GC Fuji I. These results indicate that different types of dental cement differ extensively in their genotoxic and cytotoxic potential and their ability to affect chromosomal integrity, cell-cycle progression, DNA replication and repair. Although these results cannot be directly extrapolated to the clinical situation, the potential occurrence of adverse effects caused by the RM-GICs and RCs tested in this study should be considered when making a clinical decision about dental cements.

Solid variant of a pure intracystic papillary carcinoma of the breast: case report.

Intracystic papillary carcinoma (IPC) of the breast is an uncommon malignant breast neoplasm and usually occurs in advanced age. It is characterized by a more benign behavior and a subsequent higher survival rate. We describe such a case of a 58-year-old female, who displayed a gradually growing tumor of the right breast. The lesion was well circumscribed and had a hard consistency with a cystic appearance. Mammography, breast ultrasonography and fine needle aspiration cytology failed to obtain a definite diagnosis. Based on the preoperative clinical identification of right axillary lymphadenopathy, the patient eventually underwent segmental resection of the right breast and right axillary nodal dissection. As regards the histological findings, the neoplasm corresponded to a pure intracystic papillary carcinoma of the solid variant. IPC represents a breast tumor with papillary differentiation growing inside a cyst, and excisional biopsy is often necessary to confirm the disease. Careful pathological examination is essential, to exclude the presence of coexistent ductal carcinoma in situ or invasive carcinoma.

Primary osteogenic sarcoma of the breast.

Primary osteogenic sarcomas of the breast are exceptionally uncommon. We describe such a case occurring in a 66-year-old woman who presented with a hard mass in her left breast. Mammography and breast ultrasonography showed a calcified breast lump, but features were not diagnostic. Modified radical mastectomy of the left breast, including axillary lymph node dissection, was performed. Microscopical and immunohistochemical findings established the diagnosis of primary osteogenic sarcoma. Because there was no evidence of metastasis, no further treatment was considered necessary. She remained well 15 months later, without tumour recurrence. We discuss in detail the diagnostic implications of this rare entity.

Sister-chromatid exchange, chromosomal aberrations and delays in cell-cycle kinetics in human lymphocytes induced by dental composite resin eluates.

We have investigated eluates derived from commercially available composite resin-based materials used for direct (Tetric Ceram/Ivoclar-Vivadent, Simile/Pentron, Filtek Z-250/3M ESPE) and indirect (Adoro/Ivoclar-Vivadent and Conquest Sculpture/Pentron) dental restorations, with respect to their genotoxic effects on human peripheral lymphocytes. Primary lymphocyte cultures obtained from blood samples of three healthy donors were exposed to eluates of freshly cured specimens of all the materials tested. Metaphases were induced with phytohaemagglutinin, collected after a 72-h treatment using colchicine and stained with the Fluorescence Plus Giemsa (FPG) procedure. Preparations were scored for sister-chromatid exchange (SCE) and chromosomal aberrations (CAs). The proliferation rate index (PRI) and the mitotic index (MI) were also calculated. Our results show that eluates derived from the three direct composites (Filtek Z-250, Simile and Tetric Ceram) increased the frequencies of SCE and CAs and markedly reduced PRI and MI. Tetric Ceram's eluate, being the most genotoxic of all eluates tested, increased the frequencies of SCE up to 24.40 per cell (control, 9.87 per cell) and of CAs up to 424 per 100 metaphases scored (control, 5). Moreover, it caused a pronounced decrease of the PRI down to 1.31 (control, 2.44) and of the MI down to 9.8 per thousand (control, 19.2 per thousand). In contrast, eluates derived from the laboratory-processed composites (Adoro and Conquest Sculpture) induced much less cytogenetic damage. Overall, the degree of genotoxicity and cytotoxicity decreased as follows: Tetric Ceram>Filtek Z-250>Simile>Adoro=Conquest Sculpture. These results indicate that composite resins used for direct and indirect dental restorations differ extensively in their cytotoxic and genotoxic potential and in their ability to affect chromosomal integrity, cell-cycle progression, DNA replication and repair. This underlines the impact of improved polymerization with respect to their biological behavior.

Cloning, sequencing and expression of a cDNA encoding the mouse L35a ribosomal protein during differentiation of murine erythroleukemia (MEL) cells.

In a previous study we reported that ribosomal protein S5 gene is suppressed in differentiating and not in proliferating or apoptotic murine erythroleukaemia (MEL) cells (Vizirianakis et al., 1999). We wish to report here the isolation, characterisation and expression of the full length cDNA for another ribosomal protein, the L35a (rpL35a), in MEL cells. This cDNA shares significant structural homology in both DNA and protein levels to genes encoding the rat and human L35a ribosomal proteins. Northern blot hybridisation analysis has shown that the steady-state level of rpL35a mRNA is progressively reduced during differentiation of MEL cells along the erythrocytic maturation pathway induced by DMSO or UDP-4, two structurally unrelated inducers of differentiation. However, in cells where differentiation was inhibited by N(6)-methyladenosine, the level of rpL35a RNA transcripts was not affected. In addition, rpL35a gene expression was not altered in apoptotic MEL cells. Furthermore, the suppression of L35a gene was not correlated to any change in DNA methylation at CCGG sites located at the rpL35a gene locus in undifferentiated and differentiated MEL cells, as we observed for the rpS5 gene. Overall, these data suggest that the expression of ribosomal genes, the L35a of 60S ribosomal subunit and the S5 of 40S ribosomal subunit, are regulated by a common mechanism in differentiating MEL cells, leading to the observed decrease in ribosomal function.

Protein secretion biotechnology using Streptomyces lividans: large-scale production of functional trimeric tumor necrosis factor alpha.

We evaluated the feasibility of large-scale production of biopharmaceuticals expressed as heterologous polypeptides from the Gram-positive bacterium Streptomyces lividans. As a model protein we used murine tumor necrosis factor alpha (mTNFalpha). mTNFalpha fused C-terminally to the secretory signal peptide of the subtilisin-inhibitor protein from Streptomyces venezuelae. Under appropriate fermentation conditions, significant amounts of mature mTNFalpha (80-120 mg/L) can be recovered from spent growth media. Efficient downstream processing allowing rapid purification of mTNFalpha from culture supernatants was developed. Importantly, the protein is recovered from the spent growth medium in its native trimeric state as judged by biophysical analysis. Further, mTNFalpha secreted by S. lividans is significantly more active in an in vitro apoptosis tissue culture assay than a corresponding polypeptide produced in Escherichia coli. This pilot study provides the first validation of S. lividans protein secretion as an alternative bioprocess for large-scale production of oligomeric proteins of potential therapeutic value.

Transfection of MCF-7 carcinoma cells with human integrin alpha7 cDNA promotes adhesion to laminin.

The laminin-binding alpha7beta1 integrin receptor is highly expressed by skeletal and cardiac muscles, and has been suggested to be a crucial molecule during myogenic cell migration and differentiation. Absence of integrin alpha7 subunit contributes to a form of muscular dystrophy in integrin alpha7 null mice, whereas specific mutations in the alpha7 gene are associated in humans with congenital myopathy. To examine in more detail the potential role of integrin alpha7 in human-related muscular disorders, we cloned alpha7 cDNA by RT-PCR from human skeletal muscle mRNA and then expressed the full-length human integrin alpha7 cDNA by transfection in several cell lines including MCF-7, COS-7, and NIH3T3 cells. The isolated cDNA corresponds to the human alpha7X2B alternative splice form. Expression of human alpha7 was further confirmed by transfection of chimeric human/mouse alpha7 cDNA constructs. To demonstrate the functionality of expressed human alpha7, adhesion experiments with transfected MCF-7 cells have confirmed the specific binding of human alpha7 to laminin. In addition, mouse polyclonal and monoclonal antibodies were generated against the extracellular domain of human alpha7 and used to analyze by flow cytometry MCF-7 and NIH3T3 cells transfected with the full-length of human alpha7 cDNA. These results show for the first time the exogenous expression of functional full-length human alpha7 cDNA, as well as the development of monoclonal antibodies against the human alpha7 extracellular domain. Antibodies developed will be useful for further analysis of human disorders involving alpha7 dysfunction and facilitate isolation of muscle stem cells (satellite cells) and thereby expand the opportunities for genetically modified transplantation treatment of human disease.

Transcriptional activation of prion protein gene in growth-arrested and differentiated mouse erythroleukemia and human neoplastic cells.

The prion protein (PrP) is a GPI-anchored sialoglycoprotein that has attracted worldwide attention over the years due to its involvement in the pathogenesis of transmissible spongiform encephalopathies in sheep (scrapie), cattle (BSE), and humans (CJD). To understand the precise role of the Prn-p gene in cell growth and differentiation we investigated the expression pattern of the Prn-p gene in proliferating cells and in cells arrested in growth either by confluency or by induction of terminal differentiation. Viral-transformed mouse spleen hematopoietic cells named murine erythroleukemia (MEL) and other types of inducible cells (human neuroectodermal RD/TE-671, myoid RD cells) were employed. Cells grown exponentially, at confluency, or irreversibly arrested in growth at terminal differentiation state were analyzed by fluorescence cell sorting and Northern blot hybridization to estimate the steady-state level of PrP mRNA at different phases of the cell cycle. MEL cells that failed to differentiate from treatment with N(6)-methyladenosine (N(6)mAdo), an inhibitor of differentiation, were also analyzed for PrP mRNA level. Our results indicate the following: (a) growth arrest of cells at G(1) phase by confluency or by induction of terminal differentiation led to increased accumulation of PrP mRNA transcripts, an event observed also in differentiated MEL, RD/TE-671, and RD cells independent of the inducer used; (b) treatment of MEL cells with N(6)mAdo prevented early activation of the Prn-p gene in cells treated with the inducer; and (c) cell-free nuclear run-off studies showed enhanced expression of the Prn-p gene due to transcriptional activation. These findings indicate, for the first time, that the Prn-p gene, which is thought to be a housekeeping gene, is transcriptionally activated in G(1) phase in confluent and terminally differentiated cells. This information may be valuable in understanding the overaccumulation of PrP in some differentiated tissues and may let us repress Prn-p gene activation by novel agents.

Structural and functional impairment of mitochondria in adriamycin-induced cardiomyopathy in mice: suppression of cytochrome c oxidase II gene expression.

The use of adriamycin (ADR) in cancer chemotherapy has been limited due to its cumulative cardiovascular toxicity. Earlier observations that ADR interacts with mitochondrial cytochrome c oxidase (COX) and suppresses its enzyme activity led us to investigate ADR's action on the cardiovascular functions and heart mitochondrial morphology in Balb-c mice i.p. treated with ADR for several weeks. At various times during treatment, the animals were assessed for cardiovascular functions by electrocardiography and for heart tissue damage by electron microscopy. In parallel, total RNA was extracted from samples of dissected heart and analyzed by Northern blot hybridization to determine the steady-state level of three RNA transcripts encoded by the COXII, COXIII, and COXIV genes. Similarly, samples obtained from the liver of the same animals were analyzed for comparative studies. Our results indicated that 1) treatment of mice with ADR caused cardiovascular arrhythmias characterized by bradycardia, extension of ventricular depolarization time (tQRS), and failure of QRS at high concentrations (10-14 mg/kg body weight cumulative dose); 2) the heart mitochondria underwent swelling, fusion, dissolution, and/or disruption of mitochondrial cristae after several weeks of treatment. Such abnormalities were not observed in the mitochondria of liver tissue; and 3) among the three genes of COX enzyme examined, only COXII gene expression was suppressed by ADR treatment, mainly after 8 weeks in both heart and liver. Knowing that heart mitochondria represent almost 40% of heart muscle by weight, we conclude that the deteriorating effects of ADR on cardiovascular function involve mitochondrial structural and functional impairment.

Expression of ribosomal protein S5 cloned gene during differentiation and apoptosis in murine erythroleukemia (MEL) cells.

Murine erythroleukemia (MEL) cells have been used as a suitable model system for studying cellular and molecular mechanisms of erythroid differentiation. In an effort to isolate and characterize genes whose expression change during differentiation, we cloned and sequenced a cDNA of 715 bp (rpS5) from a MEL cDNA library. The cloned mouse cDNA showed significant degree of structural homology in both DNA and protein level to known human and rat genes that encode the S5 proteins of 40S ribosomal subunit. The use of 715-bp cDNA as probe revealed the presence of an RNA transcript in the cytoplasm of MEL and human neuroectodermal RD/TE-671 cells. The steady-state accumulation level of this RNA transcript decreased upon induction of differentiation of both cell lines by treatment with DMSO and UDP-4, two structurally different inducers. Blockade of MEL cell differentiation by the inhibitor N6-methyladenosine preserved the constitutive expression of the rpS5 gene. DNA methylation analysis at CCGG sites located at the rpS5 gene locus in undifferentiated and differentiated MEL cells revealed that the suppression of the rpS5 gene during MEL cell differentiation is not related to any change in methylation at these sites. Moreover, the rpS5 gene continued to be expressed in cells undergoing serum-deprived apoptosis, like in control untreated cells. Therefore, we conclude that there may be a disparate pattern of expression of the rpS5 gene in differentiating and apoptotic cells. These data can be valuable in understanding the role of ribosomal proteins during differentiation and cell death (apoptosis) of neoplastic cells, although there is no experimental evidence that the suppression of the rpS5 gene is related mechanistically to the induction of differentiation. It may well be considered as part of the differentiation process.

Effects of hemin on apoptosis, suppression of cytochrome c oxidase gene expression, and bone-marrow toxicity induced by doxorubicin (adriamycin).

We have shown that hemin (iron-protoporphyrin IX) selectively counteracts doxorubicin (Adriamycin, ADR)-induced cytotoxicity on human leukemia K-562 cells by preventing ADR from inhibiting mitochondrial cytochrome c oxidase (COX), a novel target site for anthracyclines. Here, we investigated whether or not (a) treatment with ADR promotes apoptosis and represses the expression of two COX genes (one nuclear and one mitochondrial) in human K-562 cells in the absence and presence of hemin, and (b) injection of hemin preserves bone-marrow cellularity in ADR-myelosuppressed rats. Cultured K-562 cells were incubated with varying concentrations of ADR.HCl (0.2 microM to 5 microM) in the presence and absence of hemin (30 microM) and assessed for DNA degradation, as well as for expression of mitochondrial COXII and nuclear COXIV genes by RNA Northern blot hybridization analysis. In parallel, we investigated whether or not hemin injected i.p. in myelosuppressed rats affected ADR-induced bone-marrow cytotoxicity. These studies have shown the following: (a) ADR caused a dose- and time-dependent DNA fragmentation, characteristic of apoptosis, in K-562 cells; (b) hemin reduced the frequency of cell death caused by ADR: this effect was specific for ADR, because hemin failed to prevent apoptosis induced by methotrexate (MTX) in these cells; (c) ADR suppressed expression of COXIV and COXII genes, and exposure of ADR-treated K-562 cells to hemin did not reverse this suppression; and (d) i.p. injection of hemin in ADR-myelosuppressed rats improved bone-marrow cellularity, promoted colony formation (CFU-GM and CFU-F), and stromal cell outgrowth; moreover, hemin increased WBC counts depressed 12 days after ADR treatment. These studies indicate that hemin is a selective inhibitor of ADR-induced apoptosis of human leukemia cells and preserves bone-marrow cellularity in rats injected with ADR.

Induction of murine erythroleukemia cell differentiation is associated with methylation and differential stability of poly(A)+ RNA transcripts.

Murine erythroleukemia (MEL) cells exposed to DMSO were assessed for their ability to methylate poly(A)+ RNA and accumulate RNA transcripts of globin and nonglobin genes (c-myc, beta-actin and MER5). Cells were pulse-labeled with L-[methyl-3H]methionine, cytoplasmic RNA was isolated, selected for poly(A)+ RNA and analyzed by HPLC chromatography for methylated nucleosides. When MEL cells were exposed to inhibitors of RNA methylation (neplanocin A, 3-deazaneplanocin A and cycloleucine) and assessed for their ability to differentiate by DMSO, accumulate RNA transcripts, produce hemoglobin, methylate poly(A)+ and poly(A)- RNA and synthesize S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH), we observed the following: (a) MEL cells treated with DMSO underwent hypermethylation in poly(A)+ RNA that preferentially occurred at the 5'-cap structures (7-methylguanosine and 2'-O-methylcytidine and 2'-O-methyluridine); (b) inducer-treated MEL cells exhibited a decrease in the intracellular level of SAH that led to a lower ratio of SAH/SAM, an event that favors methylation; and (c) treatment of MEL cells with inhibitors of RNA methylation suppressed methylation of poly(A)- and poly(A)+ RNA, reversed the ratio SAH/SAM seen in differentiated MEL cells and prevented differentiation to occur. Moreover, we observed that treatment of MEL cells with selective inhibitors of RNA methylation caused fragmentation of beta major globin and c-myc mRNAs, two RNA transcripts coded by developmentally regulated genes, while had no detectable effect on the structural integrity of poly(A)+ RNA transcripts transcribed by two housekeeping genes (beta-actin and MER5). These data indicate that induction of erythroid cell differentiation of MEL cells is associated with changes in methylation of poly(A)+ RNA and selective differential stability of RNA transcripts, two events that might be related to each other.

Expression of memory, differentiation, and repression of c-myc and p53 genes in human RD/TE-671 cells induced by a ureido-derivative of pyridine (UDP-4).

Human TE-671 cells have been used to study several aspects of neuroectodermal tumors in culture. Since the human TE-671 cell lines has been re-identified as a rhabdomyosarcoma (RD) rather than a medulloblastoma due to the presence of muscle-type nicotinic acetylcholine receptors, we re-investigated the nature of RD/TE-671 cells and characterized their differentiation induced by 2-(3-ethylureido)-6-methylpyridine (UDP-4), a potent inducer of differentiation of neoplastic cells. RD cells were also used for comparative studies. RD/TE-671 cells exposed to UDP-4 were differentiated irreversibly into postmitotic cells expressing mainly neurofilaments and, to a lesser extent, myoid proteins. In contrast to RD cells that expressed preferentially myoid and not neurofilament proteins (NFPs) upon treatment with UDP-4, differentiated RD/TE-671 cells exhibited characteristic dendritic processes and expressed NFPs (NFP68, NFP160, and NFP200), parvalbumin (calcium-binding protein), and neuron-specific enolase, as well as a small amount of vimentin and desmin. In addition, differentiated RD/TE-671 cells expressed memory for differentiation and underwent an irreversible limitation of proliferation, loss of clonogenic potential, selective repression of c-myc and p53 proto-oncogenes, and changes in cell surface architecture. Treatment of RD/ TE-671 cells with nerve growth factor or epidermal growth factor in the presence of UDP-4 did not alter the phenotype of differentiated cells, whereas co-treatment with 12-O-tetradecanoylphorbol-13-acetate and UDP-4 enhanced morphological differentiation. Therefore, we conclude that: (a) RD/TE-671 cells challenged with UDP-4 express memory to differentiate in the absence of inducer; (b) in contrast to RD cells, RD/TE-671 cells appear to be multipotent cells of neuroectodermal origin capable of differentiation into cells expressing neuronal rather than myoid proteins upon treatment with UDP-4; and (c) differentiation of RD/TE-671 cells leads to selective cessation of cell proliferation and repression of c-myc and p53 proto-oncogenes.