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E Dmitrovsky

Publications and source records attributed to E Dmitrovsky.

At least 37 records · Page 2Linked to original sources
PubMed1999

Transcriptional activation of the cyclin-dependent kinase inhibitor p21 by PML/RARalpha.

Acute promyelocytic leukemia (APL) is a result of clonal expansion of hematopoietic precursors blocked at the promyelocytic stage and is associated with a t(15;17) chromosomal translocation and the expression of the PML/RARalpha fusion protein. Treatment of APL cells with retinoic acid (RA) leads to complete remission by inducing growth arrest and differentiation of these cells into granulocytes. The cyclin-dependent kinase inhibitor p21WAF1/CIP1 may be involved in terminal differentiation associated growth arrest. We showed in this study that PML/RARalpha increased the transcription of p21WAF1/CIP1 gene and the activation was further induced by RA treatment. Deletion analysis revealed a region upstream of the p21WAF1/CIP1 promoter that is required for transactivation by PML/RARalpha. Transient transfection of PML/RARalpha in cells increased the endogenous p21WAF1/CIP1 protein levels. These results suggest that the induction of APL cells differentiation by RA may be a result of the activation of p21WAF1/CIP1 by PML/RARalpha.

Cell Extracts↗
PubMed1999

Genesis, a Winged Helix transcriptional repressor, has embryonic expression limited to the neural crest, and stimulates proliferation in vitro in a neural development model.

A novel repressor of the Winged Helix (formerly HNF-3/Forkhead) transcriptional regulatory family, termed Genesis (also called HFH2), was previously found to be exclusively expressed in primitive embryonic cell lines. In this study in situ cRNA hybridization experiments revealed that Genesis was expressed during embryogenesis only in developing neural crest cells. Its expression diminished upon their terminal differentiation into sympathetic and parasympathetic neurons. Based on that finding, Genesis was retrovirally transduced into pluripotent N-Tera-2 clone D1 (NT2/D1) teratocarcinoma cells, which are a well-described in vitro model of neural development. Retinoic acid (RA) treatment will drive these cells to differentiation toward the neuronal lineage and cause an increase in expression of the cyclin-dependent kinase inhibitor p21 protein, which leads to an inhibition in cellular proliferation. Although RA-induced expression of neuronal differentiation markers was not influenced by forced overexpression of Genesis in NT2-D1 cells, proliferation of Genesis-transduced cells continued following RA treatment. RA was unable to induce the expression of the cyclin-dependent kinase inhibitor p21 in the Genesis-transduced cells, but Go/G1 tumor suppressor p53 expression was induced normally. Therefore, Genesis may play a role in the regulation of primitive neural crest development by preventing terminal quiescence through inhibition of p21 protein expression. These data also lend evidence for the hypothesis that proliferation and differentiation pathways are not irrevocably linked, but can function independently.

Animals↗
PubMed1998

Targeting the PML/RAR alpha translocation product triggers apoptosis in promyelocytic leukemia cells.

The t(15;17) rearrangement found in acute promyelocytic leukemia (APL) yields a fusion transcript, PML/RAR alpha. PML/RAR alpha expression is linked to leukemogenesis and to clinical sensitivity to all-trans retinoic acid (RA). Paradoxically, RA treatment causes transient complete remissions in most t(15;17) APL cases. The precise roles of PML/RAR alpha in triggering leukemia or in causing a maturation block are not yet known. This study explores directly these PML/RAR alpha functions in the growth and differentiation of APL cells using a hammerhead ribozyme to target PML/RAR alpha mRNA in the NB4 APL cell line. When the PML/RAR alpha cleaving but not the non-catalytic control ribozyme is introduced into the NB4 APL cell line, PML/RAR alpha protein expression is reduced. This catalysis signals growth suppression, cytotoxicity, and apoptosis without overcoming the maturation block found in these leukemic cells. These biologic effects depend on the selective pressure used to express the ribozyme from an episomal vector. Introduction of a non-catalytic, control ribozyme into NB4 cells caused no observed phenotype due to anti-sense activities. Expression of the catalytic or non-catalytic ribozymes in control cells lacking PML/RAR alpha mRNA yielded no apparent growth or differentiation effects. Thus, use of a hammerhead ribozyme that targets PML/RAR alpha expression in APL cells reveals the anti-apoptotic function of this translocation product and demonstrates that PML/RAR alpha cleavage is insufficient to overcome the differentiation block observed in these leukemic cells. Taken together, these findings indicate that persistent PML/RAR alpha expression is required to maintain basal leukemic cell growth and point to the therapeutic potential of targeting PML/RAR alpha in APL.

Apoptosis↗
PubMed1998

Targeting of PML/RARalpha is lethal to retinoic acid-resistant promyelocytic leukemia cells.

Acute promyelocytic leukemia (APL) cells, containing the t(15;17) rearrangement, express the fusion protein, PML/RARalpha. Clinically, patients respond to all-trans retinoic acid (ATRA) through complete remissions associated with myeloid maturation of leukemic cells. This clinical ATRA response of APL is linked to PML/RARalpha expression. Unfortunately, these remissions are transient and relapsed APL is often ATRA-resistant. The role PML/RARalpha plays in the growth and maturation of these APL cells with acquired ATRA resistance has not been fully explored. This study uses an ATRA-resistant NB4 cell line (NB4-R1) to investigate the contribution of PML/RARalpha expression to ATRA resistance. Targeting of PML/RARalpha in NB4-R1 cells was undertaken using two approaches: homologous recombination and hammerhead ribozyme-mediated cleavage. Reducing PML/RARalpha protein in NB4-R1 cells rendered these cells more sensitive to ATRA. These cells were growth-inhibited in ATRA, apoptosis was induced, and there was no apparent signaling of differentiation. Sequence analysis identified a mutation in the ligand binding domain (LBD) of the RARalpha portion of PML/RARalpha. Results show that these retinoid-resistant NB4 cells require persistent PML/RARalpha expression for leukemic cell growth. Taken together, these findings can account for why these cells do not respond to ATRA and how reduction of PML/RARalpha abrogates the antiapoptotic effect it confers to these leukemic cells.

Antineoplastic Agents↗
PubMed1998

FGF4 dissociates anti-tumorigenic from differentiation signals of retinoic acid in human embryonal carcinomas.

A subset of male germ cell cancers presenting with advanced stage abundantly express the fibroblast growth factor-4 (FGF4). FGF4 expression is restricted in vitro to undifferentiated embryonal carcinomas (ECs). During induced differentiation, FGF4 expression is repressed in maturation sensitive but not resistant human ECs, suggesting FGF4 plays an important role in malignant growth or differentiation of ECs. To explore these FGF4 signals in male germ cell cancers, the multipotent human EC NTERA-2 clone D1 (NT2/D1) cell line was studied. All-trans-retinoic acid (RA)-treatment of these cells induces a neuronal phenotype and represses tumorigenicity and FGF4 expression. In contrast, RA-treatment of retinoid resistant lines derived from NT2/D1 cells failed to repress FGF4 expression. This implicated FGF4 directly in regulating human EC growth or differentiation. To evaluate further this FGF4 role, FGF4 was constitutively over-expressed in NT2/D1 cells using a CMV-driven expression vector containing the neomycin resistance gene. Three stable transfectants expressing exogenous FGF4 were studied as was a control transfectant only expressing the neomycin resistance gene. RA-treatment repressed endogenous but not exogenous FGF4 expression. RA-treatment of these transfectants induced morphologic and immunophenotypic maturation, changes in RA-regulated genes, and a G1 cell cycle arrest in a manner similar to parental NT2/D1 cells. This indicated FGF4 over-expression did not block RA-mediated differentiation. As expected, RA-treatment repressed tumorigenicity of the control transfectant after subcutaneous injection into athymic mice. Despite RA-treatment, this repressed tumorigenicity was overcome in all the transfectants over-expressing FGF4. The histopathology and neovascularization did not appreciably differ between xenograft tumors derived from FGF4 over-expressing versus control transfectants. FGF4 expression studies were extended to patient-derived germ cell tumors using total cellular RNA Northern analysis and an immunohistochemical assay developed to detect FGF4 protein expression. Germ cell tumors with EC components were significantly more likely to express FGF4 mRNA (P < or = 0.0179) than other examined germ cell tumors without EC components. Immunohistochemical results from 43 germ cell tumors demonstrated increased FGF4 expression especially in non-seminomas having EC components. Thus, FGF4 promotes directly malignant growth of cultured ECs, overcomes the antitumorigenic actions of RA, and is selectively expressed in specific histopathologic subsets of germ cell tumors. Taken together, these findings indicate how differentiation and anti-tumorigenic retinoic acid signals can be dissociated in germ cell cancer.

Antineoplastic Agents↗
PubMed1998

Specific retinoid receptors cooperate to signal growth suppression and maturation of human embryonal carcinoma cells.

This study addresses the contributions of specific retinoid receptors during all-trans-retinoic acid (RA)-mediated differentiation and growth suppression of human embryonal carcinoma cells. The pleiotropic effects of RA are mediated by retinoic acid receptors (RARs) and retinoid X receptors (RXRs), members of the nuclear receptor family of transcription factors. After RA-treatment the multipotent human embryonal carcinoma cell line NTERA-2 clone D1 (NT2/D1) displays limited proliferative potential, reduced tumorigenicity, and morphologic and immunophenotypic neuronal maturation. RARgamma over-expression in NT2/D1 cells signals mesenchymal NT2/D1 terminal differentiation while RARalpha and RARbeta do not and RARgamma overcomes retinoid resistance in an NT2/D1 clone (NT2/D1-RI) having deregulated RARgamma expression. Since RARgamma transfectants do not display neuronal maturation, this study sought to identify cooperating retinoid receptors engaged in NT2/D1 differentiation. Through gain of function experiments, this report highlights RXRbeta as playing an important role along with RARgamma in signaling differentiation of NT2/D1 cells. Stable over-expression of RXRbeta, but not RXRalpha or RXRgamma, was found to signal NT2/D1 growth suppression and to induce a non-neuronal morphology and immunophenotype. Notably, co-transfection of RARgamma and RXRbeta resulted in marked growth suppression and for the first time, expression of typical neuronal markers of NT2/D1 differentiation. To clarify the role of RXRbeta and RARgamma in this differentiation program, a modified transient fibroblast growth factor-4 (FGF4) promoter-enhancer reporter assay that reflects effective RA-mediated differentiation of NT2/D1 cells was employed. Transfection of RARgamma or RXRbeta in NT2/D1 cells augments transcriptional repression of the FGF4 reporter and RARgamma and RXRbeta co-transfection markedly repressed reporter activity, indicating the combined role of these receptors in RA-induced NT2/D1 differentiation. Taken together, these findings reveal specific retinoid receptors must cooperate to signal terminal growth suppression and maturation of NT2/D1 cells. Since the transcriptional repression of FGF4 is coupled to the effective maturation of human embryonal carcinoma cells, the described co-transfection strategy should prove useful to identify genes with positive or negative effects on the differentiation program of these tumor cells.

Carcinoma, Embryonal↗
PubMed1998

Type I transforming growth factor beta receptor maps to 9q22 and exhibits a polymorphism and a rare variant within a polyalanine tract.

In a search for mutations of the type I transforming growth factor beta receptor (TbetaR-I), we mapped the gene to 9q22 and found a common polymorphism [TbetaR-I(6A)] and a rare variant [TbetaR-I(10A)] of TbetaR-I, causing an in-frame deletion of three alanines and an in-frame insertion of one alanine, respectively, in the receptor's extracellular domain. The biological relevance of the polymorphism TbetaR-I(6A) was investigated. When TbetaR-I(6A) was transiently transfected into TbetaR-I-deficient cells, the growth-inhibitory effects of transforming growth factor beta were restored. TbetaR-I(6A) and TbetaR-I(10A) frequency were assessed in 108 tumor samples and 80 nontumor samples from patients with a diagnosis of cancer, as well as in 118 normal blood donors of comparable ethnic composition. The frequency of TbetaR-I(6A) heterozygotes was fairly similar in normal blood donors (8%), in nontumor DNA of patients with a diagnosis of cancer (10%), and in tumor samples (14%). However, the frequency of TbetaR-I(6A) homozygotes among nontumor (4%) and tumor (8%) samples obtained from patients with a diagnosis of cancer was higher than that predicted by the Hardy-Weinberg law. The clinical and biological significance of TbetaR-I(6A) homozygosity needs to be further investigated.

Acute Disease↗
PubMed1997

High telomerase activity in primary lung cancers: association with increased cell proliferation rates and advanced pathologic stage.

BACKGROUND: Telomerase enzyme activity is not detected in most normal cells, a phenomenon believed to be associated with limitations on cellular proliferation. Since this activity is detected in nearly all human tumors, including non-small-cell lung cancers, it has been suggested that telomerase activation may be coupled to acquisition of the malignant phenotype. In this study, we determined whether telomerase activity was associated with tumor pathologic stage, tumor cell proliferation rates, and clinical outcome in a cohort of patients with resected non-small-cell lung cancer for whom long-term follow-up was available. METHODS: Primary tumor specimens from 99 patients treated with surgery alone and six patients treated with surgery after chemotherapy were analyzed. Telomerase activity was measured by means of a modified Telomeric Repeat Amplification Protocol (TRAP) assay. Southern blot analysis of terminal restriction fragments was used to evaluate telomere length. Immunohistochemical analysis of Ki-67, a proliferation-associated nuclear antigen, was used to assess tumor cell proliferation. RESULTS: Telomerase activity was detected in 84 of the 99 tumors treated with surgery alone; this activity was not detected in specimens of adjacent, benign lung tissue. Telomerase was detected in only three of six tumors resected after chemotherapy. For the surgery-alone group, statistically significant positive associations were found between the level of telomerase activity and tumor stage, lymph node metastasis, pathologic TNM (tumor-node-metastasis) stage, and Ki-67 immunostaining; a statistically significant inverse association was found between telomerase activity and patient age. No statistically significant differences in telomere length were found in relation telomerase activity or pathologic stage. Telomerase activity was not found to be associated with clinical outcome in a multivariate Cox proportional hazards analysis adjusted for tumor stage and lymph node status. CONCLUSIONS: High telomerase activity is detected frequently in primary non-small-cell lung cancers that exhibit high tumor cell proliferation rates and advanced pathologic stage.

Adenocarcinoma↗
PubMed1997

Posttranslational regulation of cyclin D1 by retinoic acid: a chemoprevention mechanism.

The retinoids are reported to reduce incidence of second primary aerodigestive cancers. Mechanisms for this chemoprevention are previously linked to all-trans retinoic acid (RA) signaling growth inhibition at G1 in carcinogen-exposed immortalized human bronchial epithelial cells. This study investigated how RA suppresses human bronchial epithelial cell growth at the G1-S cell cycle transition. RA signaled growth suppression of human bronchial epithelial cells and a decline in cyclin D1 protein but not mRNA expression. Exogenous cyclin D1 protein also declined after RA treatment of transfected, immortalized human bronchial epithelial cells, suggesting that posttranslational mechanisms were active in this regulation of cyclin D1 expression. Findings were extended by showing treatment with ubiquitin-dependent proteasome inhibitors: calpain inhibitor I and lactacystin each prevented this decreased cyclin D1 protein expression, despite RA treatment. Treatment with the cysteine proteinase inhibitor, E-64, did not prevent this cyclin D1 decline. High molecular weight cyclin D1 protein species appeared after proteasome inhibitor treatments, suggesting that ubiquitinated species were present. To learn whether RA directly promoted degradation of cyclin D1 protein, studies using human bronchial epithelial cell protein extracts and in vitro-translated cyclin D1 were performed. In vitro-translated cyclin D1 degraded more rapidly when incubated with extracts from RA treated vs. untreated cells. Notably, this RA-signaled cyclin D1 proteolysis depended on the C-terminal PEST sequence, a region rich in proline (P), glutamate (E), serine (S), and threonine (T). Taken together, these data highlight RA-induced cyclin D1 proteolysis as a mechanism signaling growth inhibition at G1 active in the prevention of human bronchial epithelial cell transformation.

Bronchi↗
PubMed1997

Common defects of different retinoic acid resistant promyelocytic leukemia cells are persistent telomerase activity and nuclear body disorganization.

The acute promyelocytic leukemia (APL) t(15;17) rearrangement fuses the promyelocytic leukemia (PML) gene to the retinoic acid receptor-alpha (RAR alpha). There is expression of the chimeric transcript, PML/RAR alpha, in these APL cells. These clinical APL cases respond to the differentiation agent all-trans retinoic acid (ATRA) with complete but not durable remissions because ATRA resistance develops. The NB4 APL cell line expresses PML/RAR alpha and responds to the growth inhibitory and differentiation-inducing signals of ATRA. To identify mechanisms responsible for ATRA resistance in APL, ATRA-resistant NB4 cell lines were derived from parental NB4 cells using different strategies. These lines were resistant to the growth inhibition and differentiation effects of ATRA. ATRA-resistant cells were isolated as a de novo resistant line from parental NB4 cells (NB4-R1), following chemical mutagenization and selection in ATRA (NB4-R2), or after chronic selection in ATRA (NB4-R3). Common defects linked to this ATRA resistance were found. When cultured in ATRA, these resistant cells still express PML, RAR alpha, and PML/RAR alpha proteins. Sequence abnormalities were not detected in the RAR alpha DNA binding domains cloned from a representative RA-resistant NB4 line. In ATRA-sensitive but not ATRA-resistant NB4 cells, ATRA down-regulated retinoid X receptor-alpha (RXR alpha) expression, a known marker of ATRA response in parental NB4 cells. Notably, engineered overexpression of RXR alpha in ATRA-sensitive NB4 cells did not block ATRA-mediated growth suppression. ATRA treatment of these resistant NB4 lines did not signal a decline in telomerase activity or reorganization of PML-associated nuclear bodies, but both events occurred in ATRA-sensitive NB4 cells. These ATRA-resistant NB4 lines are not fully differentiation-defective, since monocytic maturation was induced following treatment with phorbol 12-myristate 13-acetate (PMA) and 1,25 dihydroxy vitamin D3 (vitamin D3). Notably, induced monocytic differentiation of these distinct ATRA-resistant APL lines markedly repressed telomerase activity. Thus, this study suggests that persistent telomerase activity and nuclear body disorganization are linked to ATRA resistance in APL.

Antineoplastic Agents↗
PubMed1997

Cloning of the human orphan receptor germ cell nuclear factor/retinoid receptor-related testis-associated receptor and its differential regulation during embryonal carcinoma cell differentiation.

We have cloned a cDNA encoding the full-length coding region of the human homologue of the germ cell nuclear factor (GCNF)/retinoid receptor-related testis-associated receptor (RTR), from a human testis cDNA library. The amino acid sequence of human GCNF/RTR is highly homologous to that of the mouse GCNF/RTR. The largest difference between the two homologues is a 15 amino acid deletion in the human GCNF/RTR at amino acid 47. The GCNF/RTR gene was localized on human chromosome 9. Northern blot analysis using poly(A)+ RNA from different human tissues showed that GCNF/RTR mRNA is most abundantly expressed in the testis. GCNF/RTR was also highly expressed in embryonic stem cells and embryonal carcinoma cells but repressed in its differentiated derivatives. Induction of differentiation of mouse embryonal carcinoma F9 cells and human embryonal carcinoma NTERA-2 clone D1 (NT2/D1) cells by all-trans retinoic acid was accompanied by a down-regulation of GCNF/RTR. Our observations suggest that GCNF/RTR plays a role in the control of gene expression in early embryogenesis and during spermatogenesis.

Amino Acid Sequence↗
PubMed1997

Overexpression of the epidermal growth factor receptor and its ligand transforming growth factor alpha is frequent in resectable non-small cell lung cancer but does not predict tumor progression.

The epidermal growth factor receptor (EGFR) and its ligand transforming growth factor (TGF) alpha are hypothesized to form an autocrine growth loop in non-small cell lung cancer (NSCLC) and to play an important role in tumor formation and progression. We studied the association between overexpression of EGFR, TGF-alpha, or both, and overall survival of patients with resectable NSCLC. Overexpression, defined as >20% of tumor cells staining on immunohistochemistry, was examined in 96 tumor samples from consecutive patients having resection of previously untreated, well-staged NSCLC who were then followed prospectively (median follow-up, 20.7 months). The expression of three other ligands for EGFR (epidermal growth factor, cripto, and amphiregulin) was examined by Northern analysis to determine whether they might also contribute to a potential growth stimulatory loop. Overall, survival was calculated by the method of Kaplan and Meier, and prognostic factors were compared using the log-rank test. Overexpression of EGFR only was found in 32% (31 of 96), of TGF-alpha only in 10% (10 of 96), of both EGFR and TGF-alpha in 38% (37 of 96), and of neither in 19% (19 of 96) of tumors. EGFR and TGF-alpha overexpression was observed in all tumor stages and histological types but was most frequent in squamous cell carcinoma. By univariate and multivariate analyses, only tumor stage, not histology or overexpression of EGFR, TGF-alpha, or both, had a significant impact on overall survival. No expression of epidermal growth factor or cripto was observed at the total cellular RNA level of Northern analysis in tumor or benign lung, suggesting that in NSCLC these ligands may not participate in an autocrine growth stimulatory loop with EGFR. Differential overexpression of amphiregulin in malignant versus normal lung was observed, but this expression pattern did not have a prognostic impact. Thus, EGFR and TGF-alpha overexpression is frequent in early-stage NSCLC but is not associated with a survival difference. These findings suggest that this growth factor/receptor loop is more important for lung tumor formation than for tumor progression.

Adenocarcinoma↗
PubMed1996

Interferon augments PML and PML/RAR alpha expression in normal myeloid and acute promyelocytic cells and cooperates with all-trans retinoic acid to induce maturation of a retinoid-resistant promyelocytic cell line.

The PML gene is fused to the retinoic acid receptor alpha gene (RAR alpha) in the acute promyelocytic leukemia (APL) 15; 17 translocation. PML is expressed in diverse tissues and cell lines and localized in the nucleus with a typical speckled pattern. In the bone marrow, it is preferentially expressed in myeloid cells. PML appears to be transcriptionally regulated by class I and II interferons, which raises the possibility that interferons modulate the function and growth and differentiation potential of normal myeloid cells and precursors by activating PML-dependent pathways. Similarly, interferons could act on APL cells, alone or in combination with all-trans retinoic acid (RA), especially if the PML/RAR alpha fusion transcript that results from the t(15; 17) is induced by interferon. We report here that PML is expressed at low levels or not expressed in normal circulating human monocytes, lymphocytes, and polymorphonucleate cells, but is markedly induced by interferon; that PML and PML/RAR alpha expression is augmented by interferon in the NB4 APL cell line, which carries the t(15; 17), and in APL blasts from patients; that interferon inhibits growth and survival of NB4 APL cells in cooperation with RA; that interferons alone have minimal maturation effect on NB4 cells; and, finally, that interferon gamma, but not alpha or beta, induces maturation and growth suppression of NB4 cells with de novo retinoid resistance, and partially restores RA response.

Cell Differentiation↗
PubMed1996

Inhibited transformation of immortalized human bronchial epithelial cells by retinoic acid is linked to cyclin E down-regulation.

The retinoids are reported to reduce second primary aerodigestive tract tumors in patients with prior lung or head and neck carcinomas. Yet, the optimal retinoid useful for chemoprevention and those mechanisms linked to this chemoprevention are not identified. This study reports an in vitro model for carcinogen-induced transformation of immortalized human bronchial epithelial (BEAS-2B) cells that was adapted to study the anti-carcinogenic effects of all-trans-retinoic acid (RA). Following exposure to carcinogens: cigarette smoke condensate (CSC) or N-nitrosamine-4-(methylnitrosamino)-1-(3 pyridyl)-1-butanone (NNK), BEAS-2B cells exhibited evidence of transformation. This included an increased anchorage independent growth or acquired ability to form tumors in athymic mice. This transformation was inhibited by RA as demonstrated by a lack of augmented anchorage independent growth or tumor formation in athymic mice for the cells treated with RA. The BEAS-2B cells transformed by NNK exhibited an increase in cyclin E expression which was associated with an increase in the cyclin E-Cdk2 kinase activity. Over-expression of human cyclin E by transfection shows cyclin E enhances the basal clonal growth of BEAS-2B cells. In both the parental and transformed BEAS-2B cells, RA down-regulated cyclin E protein levels which was associated with an inhibition of growth and an accumulation of cells in G1. The data reported here suggest the decline of cyclin E expression represents a potential mechanism for the RA-induced growth suppression which is linked to the anti-carcinogenic effects of RA. Thus, this study reports the adaption of an in vitro model of lung carcinogenesis suitable to test the activity of chemoprevention agents.

Adenoviridae↗
PubMed1996

Transgenic expression of PML/RARalpha impairs myelopoiesis.

The translocation found in acute promyelocytic leukemia rearranges the promyelocytic leukemia gene (PML) on chromosome 15 with the retinoic acid receptor alpha (RARalpha) on chromosome 17. This yields a fusion transcript, PML/RARalpha, a transcription factor with reported dominant negative functions in the absence of hormone. Clinical remissions induced with all-trans retinoic acid (RA) treatment in acute promyelocytic leukemia are linked to PML/RARalpha expression in leukemic cells. To evaluate the PML/RARalpha role in myelopoiesis, transgenic mice expressing PML/RARalpha were engineered. A full-length PML/RARalpha cDNA driven by the CD11b promoter was expressed in transgenic mice. Expression was confirmed in the bone marrow with a reverse transcription PCR assay. Basal total white blood cell and granulocyte counts did not appreciably differ between PML/RARalpha transgenic and control mice. Cell sorter analysis of CD11b+ bone marrow cells revealed similar CD11b+ populations in transgenic and control mice. However, in vitro clonal growth assays performed on peripheral blood from transgenic versus control mice revealed a marked reduction of myeloid progenitors, especially in those responding to granulocyte/ macrophage colony-stimulating factor. Granulocyte/macrophage colony-stimulating factor and kit ligand cotreatment did not overcome this inhibition. Impaired myelopoiesis in vivo was shown by stressing these mice with sublethal irradiation. Following irradiation, PML/RARalpha transgenic mice, as compared with controls, more rapidly depressed peripheral white blood cell and granulocyte counts. As expected, nearly all control mice (94.4%) survived irradiation, yet this irradiation was lethal to 45.8% of PML/RARalpha transgenic mice. Lethality was associated with more severe leukopenia in transgenic versus control mice. Retinoic acid treatment of irradiated PML/RARalpha mice enhanced granulocyte recovery. These data suggest that abnormal myelopoiesis due to PML/RARalpha expression is an early event in oncogenic transformation.

Animals↗
PubMed1996

Transfection with a CRIPTO anti-sense plasmid suppresses endogenous CRIPTO expression and inhibits transformation in a human embryonal carcinoma cell line.

CRIPTO is a member of the epidermal growth factor (EGF) gene family originally isolated from undifferentiated human NTERA2 clone D1 (NT2D1) multipotent embryonal carcinoma cells. Retinoic acid (RA) treatment of NT2D1 cells leads to a neuronal differentiation program and to concomitant loss of CRIPTO mRNA expression. To assess the role of CRIPTO in the control of NT2D1 cell growth or differentiation, these cells were treated with 3 anti-sense oligodeoxynucleotides complementary to the 5' end of the human CRIPTO mRNA. A dose-dependent inhibition of monolayer and soft agar growth was observed with each of these CRIPTO anti-sense oligodeoxynucleotides but not with a control oligodeoxynucleotide of random sequence or with the 3 corresponding CRIPTO sense oligodeoxynucleotides. In addition, NT2D1 cells were transfected with a recombinant expression vector containing a 918-bp coding fragment of the human CRIPTO cDNA in the 3' to 5' orientation. NT2D1 CRIPTO anti-sense transfectants exhibited a significantly reduced endogenous CRIPTO mRNA and protein, a 4- to 5-fold decrease in growth rate in monolayer and a 50-70% reduction in cloning efficiency in soft agar as compared with NT2D1 parental cells or with NT2D1 cells transfected with a plasmid containing the neomycin-resistance gene alone (NT2D1 neo cells). Finally, we examined the expression of immunophenotypic markers that are modulated during the differentiation of NT2D1 cells following RA treatment. The globoseries stage-specific embryonic antigen-3 recognized by the monoclonal antibody (MAb) SSEA-3 was expressed in 60% of undifferentiated parental NT2D1 or NT2D1 neo cells and in only 20% of NT2D1 CRIPTO anti-sense transfectants, whereas it was down-regulated in all cell lines following RA treatment. A neuroectodermal antigen recognized by the A2B5 MAb, which was not expressed in parental NT2D1, in NT2D1 neo or in CRIPTO anti-sense NT2D1 cells, was induced by RA treatment in all cell lines. Taken together, our results show that inhibition of endogenous CRIPTO expression in human embryonal carcinoma cells interferes with both transformation and differentiation.

Base Sequence↗
PubMed1996

Telomerase activity is repressed during differentiation of maturation-sensitive but not resistant human tumor cell lines.

The effects of induced differentiation on telomerase activity were examined in human acute promyelocytic leukemic (NB4) and human embryonal carcinoma (NTERA-2) cells exposed to all-trans-retinoic acid or hexamethylene bisacetamide. Retinoic acid treatment of NB4 and NTERA-2 cells, and hexamethylene bisacetamide treatment of NTERA-2 cells caused a decline in telomerase activity in differentiation-sensitive but not in resistant clones of these cell lines. Changes in telomerase activity as measured by the PCR-based telomeric repeat amplification protocol assay were noted by 24-72 h of exposure to the inducer, suggesting that its regulation may precede terminal differentiation. The degree of telomerase activity decline was greater in NB4 cells than in NTERA-2 cells, probably reflecting in part a more mature state of NB4 cells after 5 days of exposure to the inducer. Mixing of protein extracts from treated and untreated cells did not suggest the presence of diffusible telomerase inhibitors. Expression of the RNA component of telomerase was also examined in NB4 cells, and its decline correlated with the reduced telomerase activity measured by the telomeric repeat amplification protocol assay during induced differentiation of these tumor cells. Taken together, these findings indicate that telomerase is a regulated enzyme system during induced human tumor cell differentiation, showing an inverse relationship between the degree of differentiation and telomerase activity. These models will be be useful to study the regulation and role of telomerase during induced differentiation of human tumor cells.

Acetamides↗