Antisense to cyclin D1 reverses the transformed phenotype of human gastric cancer cells

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Retinoic acid inhibits the growth of human myeloma cells in vitro

Retinoic acid has been shown to induce growth inhibition in a variety of cell types including human myeloma cell lines. Bone marrow plasma cells from 31 multiple myeloma (MM) patients were cultured to investigate the activity of 13-cis-retinoic acid (cRA), all-trans-retinoic acid (tRA), interferon-α (IFN-α), interferon-γ (IFN-γ), and dexamethasone (DEX), alone or in combination, on in vitro proliferation and immunoglobulin (Ig) secretion. Both cRA and tRA inhibited proliferation: the labelling index (LI) of treated cultures/controls, was 0.47±0.05 (mean ± standard error mean, M ± SEM) P < 0.0001, and 0.67 ± 0.04 (M ± SEM), P < 0.0001, respectively. The inhibitory effect of cRA was significantly superior to tRA ( P = 0.0129) and IFN-α, similar to IFN-γ and DEX. The combinations of cRA + IFNα, tRA + IFN-γ, tRA + DEX did not show any synergistic effect on myeloma proliferation. In contrast, the combination cRA + DEX (0.29 ± 0.04, M±SEM) markedly increased the effect of both cRA and DEX used as single agents.
Ig synthesis was not significantly affected by CRA, tRA, IFN-γ and the combination tRA + IFN-γ, As expected, only IFN-α ( P 0.002) and DEX ( P 0.001) inhibited Ig production. The combinations cRA + IFN-α, cRA-DEX and tRA + DEX decreased Ig secretion to the same extent as IFN-α and DEX alone respectively. In conclusion, our data indicate that tRA and especially cRA strongly inhibited plasma cell proliferation but had no effect on Ig synthesis. The combination of cRA + DEX showed the highest degree of inhibitory activity of all cytokines. alone or in combination.     

Ectopic peptides released by a human melanoma cell line that modulate the transformed phenotype.

Cells derived from a human melanoma strain by low-serum selection in monolayer were found to be capable of growth in semisolid medium, forming colonies ranging from tight to loose or dispersed. These phenotypes were found to be stable after cloning and retesting. Examination of the biological activities of ectopic peptides produced by a clone that gives rise to loose dispersed colonies revealed the presence of a transforming growth factor beta (TGF-beta; apparent Mr 14,700) and a single TGF-alpha species produced at an unusually high concentration (2.2 ng/ml). Coeluted with the TGF-alpha at approximately 22,500 was a mitogenic activity and a previously undescribed activity capable of modulating the phenotype of induced normal rat kidney fibroblast (NRK-49F) colonies in semisolid medium. This activity can modulate the usual tight colony to express a loose or dispersed phenotype characteristic of the producer cells. Both the possible role these ectopic peptides play in the expression of the transformed phenotype by the tumor cells producing them and the possible correlation between the Mr 22,500 epidermal growth factor-like peptide released by this particular tumor line and high molecular weight epidermal growth factor-like peptides found in the urine of cancer patients are discussed.     

Retinoic acid enhances the growth of only one subpopulation of granulomonocyte precursors

The effect of various concentrations of retinoic acid on the growth of committed granulomonopoietic precursors (CFU-GM) was studied on semisolid agar cultures. Retinoic acid increased the growth of day 14 CFU-GM but had no significant effect on day 7 CFU-GM. Since the latter are believed to represent the progeny of day 14 CFU-GM it is suggested that the effect of retinoic acid is confined to the early stages of granulomonocytic differentiation.     

Retinoic acid induces matrix Gla protein gene expression in human cells.

The objective of this study was to investigate the possible regulation of the vitamin K-dependent matrix Gla (gamma-carboxyglutamic acid) protein (MGP) by retinoic acid, a regulation suggested by the recent observation that the human MGP promoter has a perfect direct repeat which is nearly identical to the retinoic acid-responsive element in the retinoic acid receptor-beta gene. We report that retinoic acid strongly increases MGP mRNA levels in all human cells tested, including osteoblasts, articular cartilage chondrocytes, and fibroblasts. In osteoblastic cells, MGP mRNA levels are increased by 25-fold at 1 microM retinoic acid and achieve half-maximal levels at 0.1 microM hormone. MGP is a small secreted protein of unknown function that is synthesized in a wide variety of vertebrate tissues. The present results suggest that part of the known actions of retinoic acid on skin, bone, cartilage, and other tissues in the human may be mediated by the stimulation of MGP synthesis and the consequent effect of increased MGP secretion on nearby target cells.     

Partial reversion of the transformed phenotype in HRAS-transfected tumorigenic cells by transfer of a human gene.

The transformed phenotype of rat FE-8 cells transfected by an activated human HRAS gene was suppressed upon fusion with normal cells. An experimental approach was developed to identify and isolate a human gene capable of suppressing the transforming activity of the HRAS oncogene in FE-8 cells. Genomic DNA from human placenta was introduced into FE-8 cells by cotransfection with the plasmid pY3 conferring hygromycin B resistance. Transfectants were selected in medium containing hygromycin B. HRAS-transformed FE-8 cells showed an increased sensitivity toward ouabain when compared to their normal counterparts. Therefore, the population of transfected hygromycin B-resistant cells was treated with ouabain to eliminate cells with a transformed phenotype. Ouabain selection resulted in a small number of cell clones exhibiting a more normal phenotype. The clones had lost the morphology of transformed cells and required anchorage for growth. The tumorigenicity of transfectants in nude mice was reduced but not completely abolished. FE-8 revertants continued to express the p21 RAS protein. Human repetitive sequences contained in the DNA of a secondary transfectant were used for isolation of the suppressor gene from reverted FE-8 cells. The cloned DNA fragment was transfected into tumorigenic FE-8 cells and conferred a partial reversion of the transformed phenotype.     

Retinoic acid is a negative regulator for the differentiation of cord blood-derived human mast cell progenitors

We examined the effects of retinoids on the human mast cell development using a serum-deprived culture system. When 10-week cultured mast cells derived from CD34(+) cord blood cells were used as target cells, both all-trans retinoic acid (ATRA) and 9-cis RA inhibited the progeny generation under stimulation with stem cell factor (SCF) in a dose-dependent manner (the number of progeny grown by SCF plus RA at 10(-7) mol/L was one tenth of the value obtained by SCF alone). The early steps in mast cell development appear to be less sensitive to RA according to the single CD34(+)c-kit(+) cord blood cell culture study. The optimal concentration of RAs also reduced the histamine concentration in the cultured mast cells (3.00 +/- 0.47 pg per cell in SCF alone, 1.44 +/- 0.18 pg per cell in SCF+ATRA, and 1.41 +/- 0.10 pg per cell in SCF+9-cis RA). RT-PCR analyses showed the expression of RARalpha, RARbeta, RXRalpha, and RXRbeta messenger ribonucleic acid (mRNA) in 10-week cultured mast cells. The addition of an RAR-selective agonist at 10(-10) mol/L to 10(-7) mol/L decreased the number of mast cells grown in SCF, whereas an RXR-selective agonist at up to 10(-8) mol/L was inactive. Among RAR subtype selective retinoids used at 10(-9) mol/L to 10(-7) mol/L, only the RARalpha agonist was equivalent to ATRA at 10(-7) mol/L in its ability to inhibit mast cell growth. Conversely, the addition of excess concentrations of a RARalpha antagonist profoundly counteracted the retinoid-mediated suppressive effects. These results suggest that RA inhibits SCF-dependent differentiation of human mast cell progenitors through a specific receptor. (Blood. 2000;95:2821-2828)     

Role of BCR/ABL gene-expression levels in determining the phenotype and imatinib sensitivity of transformed human hematopoietic cells

Increased levels of Bcr-Abl expression in chronic myelogenous leukemia (CML) cells are associated with disease progression and imatinib (IM) resistance. However, it is not clear if these associations are a direct result of elevated Bcr-Abl expression. We used a human transduction model of CML to directly investigate the role of varying Bcr-Abl expression levels in determining the phenotype and IM sensitivity of hematopoietic cells. CD34(+) cells were transduced with vectors coexpressing Bcr-Abl and GFP, and cells expressing low and high levels of GFP and Bcr-Abl (BA(lo) and BA(hi)) were selected. BA(hi) cells demonstrated enhanced activation of downstream proliferative and antiapoptotic signaling and enhanced proliferation and survival compared to BA(lo) cells. Freshly isolated BA(hi) CD34(+) cells and cell lines demonstrated increased IM-mediated growth inhibition likely reflecting Bcr-Abl dependence for growth and survival. CD34(+) cells expressing BCR/ABL kinase-mutant genes demonstrated resistance to IM-mediated inhibition of proliferation and viability, which was not enhanced by increased expression of BCR/ABL kinase-mutant genes. We conclude that Bcr-Abl overexpression results in increased proliferation and antiapoptotic signaling in CD34(+) cells, but may not play a direct role in IM resistance in progenitor cells expressing either wild-type or mutant BCR/ABL genes.     

Loss of transformed phenotype in cancer cells by overexpression of the uteroglobin gene

Uteroglobin (UG) is a multifunctional, secreted protein that has receptor-mediated functions. The human UG (hUG) gene is mapped to chromosome 11q12.2-13.1, a region frequently rearranged or deleted in many cancers. Although high levels of hUG expression are characteristic of the mucosal epithelia of many organs, hUG expression is either drastically reduced or totally absent in adenocarcinomas and in viral-transformed epithelial cells derived from the same organs. In agreement with these findings, in an ongoing study to evaluate the effects of aging on UG-knockout mice, 16/16 animals developed malignant tumors, whereas the wild-type littermates (n = 25) remained apparently healthy even after 11/2 years. In the present investigation, we sought to determine the effects of induced-expression of hUG in human cancer cells by transfecting several cell lines derived from adenocarcinomas of various organs with an hUG-cDNA construct. We demonstrate that induced hUG expression reverses at least two of the most important characteristics of the transformed phenotype (i.e., anchorage-independent growth on soft agar and extracellular matrix invasion) of only those cancer cells that also express the hUG receptor. Similarly, treatment of the nontransfected, receptor-positive adenocarcinoma cells with purified recombinant hUG yielded identical results. Taken together, these data define receptor-mediated, autocrine and paracrine pathways through which hUG reverses the transformed phenotype of cancer cells and consequently, may have tumor suppressor-like effects.     

Retinoic acid inhibition of ex vivo human immunodeficiency virus-associated apoptosis of peripheral blood cells.

T cells from human immunodeficiency virus (HIV)-infected individuals undergo spontaneous and activation-induced ex vivo apoptosis. Here we report that peripheral blood mononuclear cells (PBMCs) obtained from six HIV-infected individuals exhibited reduced ex vivo DNA fragmentation and cell death after ingestion of all-trans-retinoic acid (tRA). These effects were attenuated with continued daily RA administration, which correlated with a > 5-fold decrease in serum peak RA concentrations. Incubation of PBMCs from HIV+ individuals with tRA in vitro resulted in decreased DNA fragmentation in a subset of patients, especially those having < 500 CD4+ T cells per mm3. tRA also inhibited apoptosis of preactivated normal PBMCs induced to die by restimulation, which raises the possibility of a common mechanism between activation-induced apoptosis of activated normal PBMCs and apoptosis associated with HIV infection. Whether HIV-associated apoptosis of PBMCs, and its prevention by RA, has an impact on T-cell survival or the course of disease in patients infected with HIV will require further evaluation.     

Characterization of heterotypic adherence between transformed human lymphoblastic cells and marrow stromal cells: VCAM-1 is a ligand for one of the leukemia cell adhesion proteins.

Marrow stromal cells are important in normal myelopoiesis and support growth of leukemia/lymphoma (LL) cells in vitro. We have previously described the heterotypic adherence of a human B-lymphoblastic cell line (UTMB-460) to marrow stromal cells (MSC). We have extended these observations to a human T-lymphoblastic cell line (CEM) and characterized the heterotypic adherence of B- and T-lymphoblastic cell lines to human MSC. Electron microscopy demonstrated UTMB-460 cells were in very close apposition to the MSC, but no specific intercellular junctions were noted. Under the conditions employed, these MSC express extracellular fibronectin, collagen types I and IV, intracellular laminin, and vimentin, but no factor VIII-R antigen. In addition, the MSC had receptors for the lectin Ulex europaeus agglutinin I. UTMB-460 and CEM cells do not adhere to extracellular matrix (ECM) proteins secreted by the MSC, i.e., fibronectin, collagen types I, III, or IV, or laminin. Monoclonal antibodies (MoAbs) against CD11a, CD11b, CD18, and CD54 and a polyclonal anti-human fibronectin antibody do not inhibit attachment of either B- or T-lymphoblastic cells to MSC. Peptides GRGES and GRGDS did not inhibit adherence of UTMB-460 and CEM cells to MSC. In contrast, the anti-vascular cell adhesion molecule (VCAM)-1 MoAb (4b9) caused significant inhibition (p < 0.01) of the adherence of both UTMB-460 and CEM cells to normal human MSC monolayers. These data suggest: (1) that MSC to which lymphoblastic cells adhere are specialized mesenchymal cells; (2) that the membrane interactions between T- and B-lymphoblastic cells and MSC involve close apposition of cell membranes of MSC and the lymphoblastic cells; (3) that the heterotypic adherence between B- and T-lymphoblastic cell lines (UTMB-460 and CEM) and MSC does not involve the RGD recognition sequence of the integrin family, the B2 leukocyte integrins, CD44, LAM-1, or the ECM proteins examined; and (4) that VCAM-1 may at least be partially responsible for heterotypic adherence between human MSC and B- and T-lymphoblastic cells.     

Conversion of a radioresistant phenotype to a more sensitive one by disabling erbB receptor signaling in human cancer cells

Inhibition of cell growth and transformation can be achieved in transformed glial cells by disabling erbB receptor signaling. However, recent evidence indicates that the induction of apoptosis may underlie successful therapy of human cancers. In these studies, we examined whether disabling oncoproteins of the erbB receptor family would sensitize transformed human glial cells to the induction of genomic damage by γ-irradiation. Radioresistant human glioblastoma cells in which erbB receptor signaling was inhibited exhibited increased growth arrest and apoptosis in response to DNA damage. Apoptosis was observed after radiation in human glioma cells containing either a wild-type or mutated p53 gene product and suggested that both p53-dependent and -independent mechanisms may be responsible for the more radiosensitive phenotype. Because cells exhibiting increased radiation-induced apoptosis were also capable of growth arrest in serum-deprived conditions and in response to DNA damage, apoptotic cell death was not induced simply as a result of impaired growth arrest pathways. Notably, inhibition of erbB signaling was a more potent stimulus for the induction of apoptosis than prolonged serum deprivation. Proximal receptor interactions between erbB receptor members thus influence cell cycle checkpoint pathways activated in response to DNA damage. Disabling erbB receptors may improve the response to γ-irradiation and other cytotoxic therapies, and this approach suggests that present anticancer strategies could be optimized.     

Expression of simian virus 40 early genes in transformed rat cells is correlated with maintenance of the transformed phenotype.

Early viral polypeptides synthesized in simian virus 40 rat transformants were identified by immunoprecipitation using anti-T (tumor) antigen immune serum. Four polypeptide classes could be identified, which were not detectable in extracts of nontransformed cells and were not precipitated from transformed cell extracts by nonimmune serum. Their apparent M(r) were 92,000, 63,000, 56,000, and 19,000. A similar pattern was observed in extracts from lytically infected cells, but the relative rate of radioactive labeling of the M(r) 63,000 and 56,000 species was in this case significantly lower than in transformed cells. In tsA30 transformants of type A, which maintain the transformed phenotype at high temperature, only minor quantitative variations of this pattern were observed when the cultures were shifted from 33 degrees to 40.5 degrees . In contrast, the rate of labeling of the four virus-specific polypeptides was decreased by 90% or more at high temperature in the temperature-sensitive N transformants. In all cases, a coordinated variation of the radioactivity associated with the different polypeptide classes was observed. These results suggest that the synthesis or processing, or both, of the viral early proteins may be controlled by different mechanisms in various types of simian virus 40 transformants and, furthermore, that it may be under the positive control of a virus-coded protein in transformed cells of type N.     

Regulation of transforming growth factor-beta1 expression by granulocyte macrophage-colony-stimulating factor in leiomyoma and myometrial smooth muscle cells

Human myometrium and leiomyomas express granulocyte macrophage-colony-stimulating factor (GM-CSF), transforming growth factor-beta (TGFbeta), and their receptors. Overexpression of TGFbeta and, to a limited extent, GM-CSF has been associated with tissue fibrosis throughout the body, including leiomyomas. The objective of the present study was to determine the action of GM-CSF on leiomyoma and myometrial smooth muscle cells (LSMC and MSMC) and examine whether the action of GM-CSF is mediated through the induction of TGFbeta1 expression. Using competitive quantitative RT-PCR and enzyme-linked immunosorbent assay, we found that LSMC express significantly higher GM-CSF messenger ribonucleic acid (mRNA; 0.6 +/- 0.1 x 10(3) copies of mRNA/microg total RNA) and protein (0.75 +/- 0.2 ng/mL) than MSMC (0.5 +/- 0.1 x 10(2) copies of mRNA and 0.45 +/- 0.07 ng/mL protein; P < 0.05). In addition, LSMC expressed significantly higher TGFbeta1 mRNA (1.6 +/- 0.3 x 10(4) copies of mRNA/microg total RNA) than MSMC (2.4 +/- 0.4 x 10(3) copies) and synthesized and secreted more TGFbeta1 protein (1.7 +/- 0.2 vs. 0.5 +/- 0.02 ng/mL); whereas MSMC contained more cell-associated TGFbeta1 (56.2 +/- 1.2 ng/mL) than LSMC (35.2 +/- 1.2 ng/mL; P < 0.05). We found that GM-CSF (0.01-100 ng/mL) has limited mitogenic activity for LSMC but not for MSMC determined by the rate of [3H]thymidine incorporation and cell proliferation assay. However, GM-CSF at 1 ng/mL increased its own production, the expression of TGFbeta1 mRNA, the cell-associated TGFbeta1 protein content in both cell types, and TGFbeta1 released into the culture-conditioned medium of LSMC (P < 0.05). TGFbeta1 also increased its own mRNA and protein expression, but had no effect on cell-associated TGFbeta1 in both cell types (P < 0.05). Cotreatment of LSMC and MSMC with GM-CSF and TGFbeta1 induced changes similar to those produced by GM-CSF in both cells. In conclusion, our data suggest that GM-CSF is not a mitogen for MSMC and LSMC, but it regulates its own expression and the expression of TGFbeta1 by these cells, a regulatory interaction that may account for the GM-CSF-induced tissue fibrosis that occurs in leiomyomas.     

Retinoic acid regulates IgG Fc receptor expression in human myelomonocytic leukemia cells and normal peripheral monocytes

The regulation of IgG Fc receptor (Fc gamma R) expression by retinoic acid (RA) in human myelomonocytic cells at different stages of maturation was studied. RA suppressed IgG-coated erythrocyte (EA) rosette formation of myelomonocytic cells blocked at relatively late stages of differentiation such as ML-1, U-937, THP-1-T, normal monocytes, and fresh cells of patients with acute myelomonocytic leukemia. However, RA increased the percentage of EA rosetting promyelocytes of HL-60 and of patients with acute promyelocytic leukemia and a part of myeloblasts isolated from acute myelogenous leukemia patients. Other myeloblasts including KG-1a, KG-1, and fresh cells from patients with acute myelogenous leukemia were not affected. A kinetic study using HL-60 and THP-1-T demonstrated that an increase required at least a 48-h exposure and that the maximum decrease required approximately 6 h. The RA effect on both cell lines was dose-dependent. The number of Fc gamma R of HL-60 and THP-1-T treated with RA became very close, although untreated THP-1 had almost 10 times as many as HL-60. Kd for IgG in both THP-1-T and HL-60, either untreated or treated with RA, remained unchanged. These observations indicate that one of the important roles of RA is regulation of Fc gamma R expression in myeloid cells.     

Upregulated expression of Ezrin and invasive phenotype in malignantly transformed esophageal epithelial cells

AIM: To investigate the correlation between ezrin expression and invasive phenotype formation in malignantly transformed esophageal epithelial cells. METHODS: The experimental cell line employed in the present study was originated form the progressive induction of a human embryonic esophageal epithelial cell line (SHEE) by the E6E7 genes of human papillomavirus (HPV) type 18. The cells at the 35(th) passage after induction called SHEEIMM were in a state of immortalized phase and used as the control, while that of the 85(th) passage denominated as SHEEMT represented the status of cells that were malignantly transformed. The expression changes of ezrin and its mRNA in both cell passages were respectively analyzed by RT-PCR and Western blot. Invasive phenotype was assessed in vivo by inoculating these cells into the severe combined immunodeficient (SCID) mice via subcutaneous and intraperitoneal injection, and in vitro by inoculating them on the surface of the amnion membranes, which then was determined by light microscopy and scanning electron microscopy. RESULTS: Upregulated expression of ezrin protein and its mRNA was observed in SHEEMT compared with that in SHEEIMM cells. The SHEEMT cells inoculated in SCID mice were observed forming tumor masses in both visceral organs and soft tissues in a period of 40 days with a special propensity to invading mesentery and pancreas, but did not exhibit hepatic metastases. Pathologically, these tumor cells harboring larger nucleus, nucleolus and less cytoplasm could infiltrate and destroy adjacent tissues. In the in vitro study, the inoculated SHEEMT cells could grow in cluster on the amniotic epithelial surface and intrude into the amniotic stroma. In contrast, unrestricted growth and invasiveness were not found in SHEEIMM cells in both in vivo and in vitro experiment. CONCLUSION: The upregulated ezrin expression is one of the important factors that are possibly associated with the invasive phenotype formation in malignantly transformed esophageal epithelial cells.     

A novel selective progesterone receptor modulator asoprisnil (J867) inhibits proliferation and induces apoptosis in cultured human uterine leiomyoma cells in the absence of comparable effects on myometrial cells

CONTEXT: Asoprisnil, a selective progesterone (P4) receptor (PR) modulator (SPRM) with mixed P4 agonist/antagonist activities, reduces uterine leiomyoma volume in a dose-dependent manner in the presence of follicular phase estrogen concentrations. The evidence from clinical studies suggests that asoprisnil may directly target the uterine leiomyomata. OBJECTIVE AND METHODS: The present study evaluated the effects of asoprisnil on cell proliferation, the expression of apoptosis-related proteins, and apoptosis in cultured human uterine leiomyoma cells and matched normal myometrial cells. PR-A and PR-B expression in the two types of cells was comparatively evaluated. Cell proliferation, proliferating cell nuclear antigen (PCNA)-positive rate, and TUNEL-positive rate were assessed by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay, immunocytochemistry, and terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick end labeling (TUNEL) assay, respectively. The expression of apoptosis-related proteins and PR was assessed by Western blot analysis. RESULTS: Compared with untreated cultures, asoprisnil decreased the number of viable cultured cells, the PCNA-positive rate, and PCNA protein expression in cultured leiomyoma cells. Asoprisnil increased the TUNEL-positive rate, cleaved caspase-3, and cleaved poly(adenosine 5'-diphosphate-ribose) polymerase expression and decreased Bcl-2 protein expression in cultured leiomyoma cells. These effects were dose and time dependent. In cultured myometrial cells, however, asoprisnil did not affect cell proliferation and apoptosis. PR-B expression was elevated in cultured leiomyoma cells compared with cultured myometrial cells, whereas no differences in PR-A expression were noted between the two cell types. CONCLUSIONS: These results show that asoprisnil inhibits proliferation and induces apoptosis in cultured uterine leiomyoma cells in the absence of comparable effects on cultured normal myometrial cells, suggesting a cell type-specific effect.