Inhibited Cell Colony Formation Research Articles

Selective inhibitory effects of artemisinin and holotransferrin on human hepatocellular carcinoma SMMC-7721 cells

Objective To investigate the selective inhibitory effect of artemisinin and holotransferrin on human hepatoeellular carcinoma SMMC-7721 cells in vitro.Methods MTT and trypan blue assay were used to assess the proliferation and viability of SMMC-7721 eeUs treated with artemisinin and holotransferrin.The inhibition of colony formation elicited by artemisinin was determined by colony formation assay.Results Artemisinin significantly inhibited SMMC-7721 cell growth,colony formation and cell viability at various concentrations (P < 0.05).It was found that combined incubation with holotransferrin, which increased the concentration of ferrous iron in cancer cells, sensitized the growth inhibitory effect of artemisinin on SMMC-7721 cells (P < 0.01).Conclusion Artemisinin and holotransferrin effectively inhibited SMMC-7721 cell viability, growth, and proliferation, which may provide a potential therapeutic choice for liver cancer. Key words: Carcinoma, hepatoeellular; Artemisinin; Transferrin; Cell growth; Cell via-bility

Differential Effect of Imatinib and Synergism of Combination Treatment with Chemotherapeutic Agents in Malignant Glioma Cells

Imatinib mesylate (STI571, Gleevec) is a signal transduction inhibitor and novel anti-cancer agent. It selectively inhibits aberrantly activated tyrosine kinases in malignant cells, for example, bcr-abl in leukaemia, platelet-derived growth factor receptor and stem cell factor receptor (c-Kit) in solid cancers including malignant glioma. However, recently published clinical studies with imatinib monotherapy in patients with malignant glioma demonstrated only very modest anti-tumour activity. The aim of this study was to investigate the biological activity of imatinib, its cellular mechanisms of action and its synergism with other chemotherapeutic agents in human malignant glioma cells in culture. Expression of PDGF/R and c-Kit was analyzed by RT-PCR. Proliferation was measured by MTT assays and drug synergy was assessed by the Chou-Talalay method. Cell cycle and apoptosis were analyzed by flow cytometry and migration by monolayer migration assays. Multi-immunoblot was performed on imatinib-treated and control malignant glioma cells. Results indicate that imatinib is more effective in inhibiting cell colony formation and migration rather than proliferation. Imatinib treatment caused cell cycle arrest of glioma cells in G0-G1 or G2/M, with significant elevation of a few cyclin-dependent kinases. Furthermore, imatinib acted synergistically with chemotherapy agents, such as the DNA alkylating agent, temozolomide, and riboneucleotide reductase inhibitors, for example, hydroxyurea at varied effective dose levels. In conclusion, imatinib exerts varied biological effects on malignant glioma cells in culture. Synergistic interaction of imatinib with chemotherapy agents may be related to cell cycle control mechanisms and could be potentially important in a clinical setting.

Identification of MSRA gene on chromosome 8p as a candidate metastasis suppressor for human hepatitis B virus-positive hepatocellular carcinoma

BackgroundThe prognosis of patients with hepatocellular carcinoma (HCC) still remains very dismal, which is mainly due to metastasis. In our previous studies, we found that chromosome 8p deletions might contribute to metastasis of HCC. In this study, we aimed to identify the candidate metastatic suppressor gene on chromosome 8p.MethodsOligo-nucleotide microarrays which included 322 genes on human chromosome 8p were constructed to analyze the difference in gene expression profiles between HCC tissues with and without metastasis. The leading differentially expressed genes were identified and selected for further analysis by real-time PCR and Western blotting. Recombinant expression plasmid vectors for each target gene were constructed and transfected into HCC cells and its in vitro effects on proliferation and invasion of HCC cells were also investigated.ResultsSixteen leading differentially expressed genes were identified from the HCC tissues with metastasis compared with those without metastasis (p < 0.01, q < 16 %). Among of the 10 significantly down-regulated genes in HCC with metastasis, methionine sulfoxide reductase A (MSRA) had the lowest p value and false discovery rate (FDR), and was considered as a potential candidate for metastasis suppressor gene. Real-time PCR and Western blotting confirmed that the mRNA and protein expression levels of MSRA were significantly decreased in HCC with metastasis compared with those without metastasis (p < 0.001), and MSRA mRNA level in HCCLM6 cells (with high metastatic potential) was also much lower than that of other HCC cell lines. Transfection of a recombinant expression plasmid vector and overexpression of MSRA gene could obviously inhibit cell colony formation (4.33 ± 2.92 vs. 9.17 ± 3.38, p = 0.008) and invasion (7.40 ± 1.67 vs. 17.20 ± 2.59, p= 0.0001) of HCCLM6 cell line.ConclusionMSRA gene on chromosome 8p might possess metastasis suppressor activity in HCC.

Quantitative hypermethylation of NMDAR2B in human gastric cancer

NMDA receptor Type 2B (NMDAR2B) is a candidate TSG first identified in esophageal squamous cell carcinoma (ESCC). To evaluate NMDAR2B methylation in gastric cancer progression, we performed quantitative methylation-specific PCR (MSP), RT-PCR and immnunohistochemistry (IHC) in primary gastric tissues and colony formation assays in gastric cancer cell lines. We found that the expression of NMDAR2B was reactivated by the demethylating agent, 5-aza-2'-deoxycytidine, with or without trichostatin A in gastric cancer cell lines. Moreover, inactivation of NMDAR2B was found to be closely correlated with promoter methylation status in gastric cell lines and primary gastric tumors. IHC data also showed that NMDAR2B was specifically expressed in gastric epithelial cells and its expression was diminished or absent in gastric cancer epithelium. Quantitative analysis of NMDAR2B promoter methylation showed 61% (17/28) hypermethylation in primary gastric tumors versus 5% (1/20) in normal gastric tissues from nongastric cancer patients. Forced over-expression of NMDAR2B in gastric cancer cell lines significantly inhibited cell colony formation. Taken together, the above results suggest that NMDAR2B methylation is a common and important biologically relevant event in gastric cancer progression.

Normal bone marrow hematopoietic stem cell reserves and normal stromal cell function support the use of autologous stem cell transplantation in patients with multiple sclerosis

Bone marrow (BM) stem cell reserves and function and stromal cell hematopoiesis supporting capacity were evaluated in 15 patients with multiple sclerosis (MS) and 61 normal controls using flow cytometry, clonogenic assays, long-term BM cultures (LTBMCs) and enzyme-linked immunosorbent assays. MS patients displayed normal CD34+ cell numbers but a low frequency of colony-forming cells (CFCs) in both BM mononuclear and purified CD34+ cell fractions, compared to controls. Patients had increased proportions of activated BM CD3+/HLA-DR+ and CD3+/CD38+ T cells that correlated inversely with CFC numbers. Patient BM CD3+ T cells inhibited colony formation by normal CD34+ cells and patient CFC numbers increased significantly following immunomagnetic removal of T cells from BMMCs, suggesting that activated T cells may be involved in the defective clonogenic potential of hematopoietic progenitors. Patient BM stromal cells displayed normal hematopoiesis supporting capacity indicated by the CFC number in the nonadherent cell fraction of LTBMCs recharged with normal CD34+ cells. Culture supernatants displayed normal stromal derived factor-1 and stem cell factor/kit ligand but increased flt-3 ligand levels. These findings provide support for the use of autologous stem cell transplantation in MS patients. The low clonogenic potential of BM hematopoietic progenitors probably reflects the presence of activated T cells rather than an intrinsic defect.

Shared Epigenetic Mechanisms in Human and Mouse Gliomas Inactivate Expression of the Growth SuppressorSLC5A8

Tumors arise in part from the deleterious effects of genetic and epigenetic mechanisms on gene expression. In several mouse models of human tumors, the tumorigenic phenotype is reversible, suggesting that epigenetic mechanisms also contribute significantly to tumorigenesis in mice. It is not known whether these are the same epigenetic mechanisms in human and mouse tumors or whether they affect homologous genes. Using an integrated approach for genome-wide methylation and copy number analyses, we identified SLC5A8 on chromosome 12q23.1 that was affected frequently by aberrant methylation in human astrocytomas and oligodendrogliomas. SLC5A8 encodes a sodium monocarboxylate cotransporter that was highly expressed in normal brain but was significant down-regulated in primary gliomas. Bisulfite sequencing analysis showed that the CpG island was unmethylated in normal brain but frequently localized methylated in brain tumors, consistent with the tumor-specific loss of gene expression. In glioma cell lines, SLC5A8 expression was also suppressed but could be reactivated with a methylation inhibitor. Expression of exogenous SLC5A8 in LN229 and LN443 glioma cells inhibited colony formation, suggesting that it may function as a growth suppressor in normal brain cells. Remarkably, 9 of 10 murine oligodendroglial tumors (from p53+/- or ink4a/arf+/- animals transgenic for S100beta-v-erbB) showed a similar tumor-specific down-regulation of mSLC5A8, the highly conserved mouse homologue. Taken together, these data suggest that SLC5A8 functions as a growth suppressor gene in vitro and that it is silenced frequently by epigenetic mechanisms in primary gliomas. The shared epigenetic inactivation of mSLC5A8 in mouse gliomas indicates an additional degree of commonality in the origin and/or pathway to tumorigenesis between primary human tumors and these mouse models of gliomas.

Mda-5: An interferon-inducible putative RNA helicase with double-stranded RNA-dependent ATPase activity and melanoma growth-suppressive properties.

Human melanoma cells can be reprogrammed to terminally differentiate and irreversibly lose proliferative capacity by appropriate pharmacological manipulation. Subtraction hybridization identified melanoma differentiation-associated gene-5 (mda-5) as a gene induced during differentiation, cancer reversion, and programmed cell death (apoptosis). This gene contains both a caspase recruitment domain and putative DExH group RNA helicase domains. Atypical helicase motifs of MDA-5 deviate from consensus sequences but are well conserved in a potentially new group of cloned and hypothetical proteins. mda-5 is an early response gene inducible by IFN and tumor necrosis factor-alpha, responding predominantly to IFN-beta. Protein kinase C activation by mezerein further augments mda-5 expression induced by IFN-beta. Expression of mda-5 is controlled transcriptionally by IFN-beta, and the MDA-5 protein localizes in the cytoplasm. mda-5 displays RNA-dependent ATPase activity, and ectopic expression of mda-5 in human melanoma cells inhibits colony formation. In these contexts, mda-5 may function as a mediator of IFN-induced growth inhibition and/or apoptosis. MDA-5 is a double-stranded RNA-dependent ATPase that contains both a caspase recruitment domain and RNA helicase motifs, with a confirmed association with growth and differentiation in human melanoma cells.

Human cytomegalovirus induced inhibition of hematopoietic cell line growth is initiated by events taking place before translation of viral gene products.

Bone marrow suppression with leukopenia is frequently observed during human cytomegalovirus (HCMV) infection, and in vitro the cell colony formation of bone marrow progenitors is directly inhibited by HCMV. To better understand the mechanisms of HCMV's ability to directly inhibit the cell colony formation of hematopoietic cells, we examined the effect of HCMV infection on four hematopoietic cell lines, ML-3, HL-60, KG-1, and U-937. Similarly to the observed effect on hematopoietic progenitors, HCMV significantly inhibited the cell colony formation of KG-1 and U-937 cells, 40% and 30% respectively. Following HCMV infection, uptake of HCMV pp65 was detected in all cell lines. In contrast, no immediate early protein production could be observed. When the cell line KG-1 was challenged with UV-inactivated HCMV or with HCMV dense bodies, containing pp65 and other matrix proteins, a 20% to 25% inhibition of cell colony formation was found. In addition, a dose-dependent inhibition of proliferation of the KG-1 cells challenged with intact or UV-inactivated HCMV, was observed. Transfection of this cell line with vectors containing genes for the HCMV matrix protein pp65, revealed no inhibitory effect. In contrast, the transfection with pp71 resulted in a 20% growth inhibition. These results indicate that HCMV can induce inhibition of cell colony formation of hematopoietic cells without transcription of HCMV regulatory proteins, and that at least one HCMV matrix protein may play an important role in this inhibitory effect.

Ei24, a p53 response gene involved in growth suppression and apoptosis.

DNA damage and/or hyperproliferative signals activate the wild-type p53 tumor suppressor protein, which induces a G(1) cell cycle arrest or apoptosis. Although the mechanism of p53-mediated cell cycle arrest is fairly well defined, the p53-dependent pathway regulating apoptosis is poorly understood. Here we report the functional characterization of murine ei24 (also known as PIG8), a gene directly regulated by p53, whose overexpression negatively controls cell growth and induces apoptotic cell death. Ectopic ei24 expression markedly inhibits cell colony formation, induces the morphological features of apoptosis, and reduces the number of beta-galactosidase-marked cells, which is efficiently blocked by coexpression of Bcl-X(L). The ei24/PIG8 gene is localized on human chromosome 11q23, a region frequently altered in human cancers. These results suggest that ei24 may play an important role in negative cell growth control by functioning as an apoptotic effector of p53 tumor suppressor activities.

P202 self-associates through a sequence conserved among the members of the 200-family proteins

Murine p202 is an interferon-inducible primarily nuclear phosphoprotein (52 kDa) whose expression in transfected cells inhibits colony formation. p202-binding proteins include the pocket proteins (pRb, p107 and p130), a p53-binding protein (sm53BP1), and transcription factors (e.g. NF-κB (p50 and p65), AP-1 (c-Fos and c-Jun), E2F-1, E2F-4, MyoD, and myogenin). p202 modulates the transcriptional activity of these factors in transfected cells. Here we demonstrate that p202 self-associates directly and a sequence in p202, which is conserved among the members of the 200-family proteins, was sufficient for self-association in vitro. Our observations reported herein raise the possibility that self-association of p202 may provide a mechanism for the regulation of its activity.

P202 Prevents Apoptosis in Murine AKR-2B Fibroblasts

p202 is an interferon (IFN)-inducible, primarily nuclear, phosphoprotein (52-kDa) whose overexpression in transfected cells inhibits colony formation. p202 binds to the retinoblastoma tumor suppressor protein and two other members of the pocket family proteins (p107 and p130). Moreover, overexpression of p202 in transfected cells inhibits the transcriptional activity of E2Fs (E2F-1/DP-1 and E2F-4/DP-1), p53, AP-1 c-Fos and c-Jun, NF-κB p50 and p65. Here we demonstrate that inhibition of endogenous p202 production in murine AKR-2B fibroblasts did not result in an increase in cell proliferation. Instead, these cells exhibited increased susceptibility to apoptosis in response to decrease in serum concentrations in the growth medium. These observations are consistent with the notion that normal levels of p202 may be needed for the regulation of cell proliferation.

Retinoic acid nuclear receptor β inhibits breast carcinoma anchorage independent growth

Retinoids modulate cellular proliferation and mediate gene function through a series of nuclear receptors. The retinoic acid nuclear receptor beta (RAR beta) plays an important role in the differentiation of a number of cell types. We now demonstrate that RAR beta expression is confined to normal mammary tissue and is not expressed in either immortalized normal or malignant cell lines. Treatment of RAR beta-transfected MDA-MB-231 cells with 1 microM all-trans-retinoic acid (RA) significantly inhibited monolayer growth of the cells which express recombinant RAR beta. RAR beta-expressing MDA-MB-231 cells formed significantly smaller and fewer colonies in soft agar than the mock-transfected cells. Addition of 1 microM RA stimulated colony size and number in the RAR beta-transfected MDA-MB-231 cells. In contrast to the RAR beta-expressing cells, colony formation by the RAR alpha-expressing cells was similar to the mock-transfected controls and the addition of 1 microM RA to the RAR alpha-transfected cells inhibited colony formation. While demonstrating decreased colony formation in agar, RAR beta-expressing MDA-MB-231 cells failed to exhibit decreased growth in SCID mice. Our results show that RAR beta functions as a negative regulator of growth in breast epithelial cells. In addition, the growth of these cells is differentially regulated by RAR alpha and RAR beta which is most likely the result of the modulation of different genes.

High-Affinity and Low-Affinity CCK A Receptor States Mediate Specific Growth-Inhibitory Effects on CHO Cells

To relate specific effects on growth and transformation to activation of specific affinity states of the CCK A receptor stably expressed in CHO cells we compared responses to the CCK analogues JMV-180 and CCK 8. CCK 8 led to an inhibition of both cell proliferation and transformation. Effects on proliferation were indicated by a reduction of DNA synthesis and cell numbers. Effects on transformation were indicated by a reduction of colony formation in soft-agar. JMV-180 did not inhibit cell proliferation although a small inhibitory effect on DNA synthesis was observed. JMV-180 inhibited the maximal effects of CCK 8 on cell proliferation and DNA synthesis. In contrast, JMV-180 substantially inhibited cell colony formation in soft-agar and did not inhibit the effects of CCK 8 on this parameter. Collectively these data with receptor affinity state specific analogues indicated that inhibition of cell proliferation and growth in soft-agar can be attributed to activation of distinct affinity states. Thus, different second messengers are likely responsible for the inhibitory effects on anchorage-dependent and -independent growth.

Selenite-induced inhibition of colony formation by buthionine sulfoximine-sensitive and resistant cell lines.

We previously demonstrated that treatment of HeLa cells with buthionine sulfoximine (BSO), which decreases the level of cellular glutathione, resulted in a decrease in the potency of selenite in inhibiting cell colony formation. We have now examined the effect of selenite on normal human lung fibroblast (CCL-210) cells, which resemble HeLa cells in their sensitivity to BSO, and on human lung adenocarcinoma (A549) cells, which are relatively insensitive to BSO. We have found that BSO treatment caused an approximately fourfold decrease in selenite potency in the CCL-210 cells, but had no significant effect on its potency in A549 cells. These results support the hypothesis that for selenite to exert its cytotoxic effect, it must undergo the reaction with an SH compound to form the selenotrisulfide. As a result of the lower sensitivity of the tumor cells to BSO, it was possible to achieve a large differential sensitivity to the cytotoxic effect of selenite.

Inhibition of cellular thioredoxin reductase by diaziquone and doxorubicin: Relationship to the inhibition of cell proliferation and decreased ribonucleotide reductase activity

The flavoenzyme thioredoxin reductase (TR) is an important enzyme for many aspects of cellular function. The antitmnor quinones diaziquone and doxorubicin have been shown to produce a time- and concentration-dependent inhibition of TR when incubated for up to 24 hr with intact A204 human rhabdomyosarcoma cells. There was a positive correlation between the inhibition of TR and the inhibition of cell colony formation measured 7 days later for diaziquone ( r = 0.84, P < 0.01), and for doxorubicin ( r=0.87, P < 0.01). 2,6-Dichloroindophenol, which in previous studies was shown to be a good inhibitor of TR in vitro, was a poor inhibitor of TR in intact A204 cells and there was no significant correlation with inhibition of colony formation. The activity of ribonucleotide reductase, which catalyzes the first unique step of DNA synthesis and which obtains its reducing equivalents from TR through thioredoxin, was decreased in diaziquone- and doxorubicin treated A204 cells. We suggest that the inhibition of TR by some antitumor quinones leading to a decreased activity of TR and, consequently, a decreased activity of thioredoxin-dependent enzymes including ribonucleotide reductase may contribute to the growth inhibitory activity of these quinones.

Inhibitory activity of interleukin 2-activated lymphocytes on human granulocyte precursors (CFU-g).

Interleukin 2-activated lymphocytes (lymphokine-activated killer [LAK] cells) cultured from 2 to 14 days were added to the cultures of granulocyte precursors (CFU-g). The LAK cells inhibited colony formation of granulocyte precursors; LAK cells cultured for five days showed the strongest inhibitory activity on colony formation. The presence of cell-to-cell interaction between LAK cells and bone marrow mononuclear cells (BMNC) in CFU-g assays emphasized the LAK cell-derived colony inhibitory activity (LAK-CIA), but cell-to-cell interaction was not always a requirement for LAK-CIA, since LAK cells were also found to inhibit colony formation without such interaction. This report shows that LAK cells can inhibit in vitro colony formation of granulocyte precursors. We therefore concluded that the observed CIA is caused by soluble factor(s) derived from LAK cells, and that E-rosette-forming cells are manifesting LAK-CIA.

Calcium dependence of chemical carcinogen induced morphological transformation of Syrian hamster embryo cells

The effect of extracellular calcium upon carcinogen induced morphological transformation was evaluated in Syrian hamster embryo cells. Reduction in [Ca 2+] from 1.8 mM to 0.2 mM throughout the 6 days between exposure of the cells to 2.5 μg benzo[ a]pyrene (BP)/ml and examination of the cells for transformation inhibited both cell proliferation and transformation as measured by the frequencies of colony formation and morphological transformation. The transformation frequency among surviving cells, i.e. frequency/cell colony, however, was nearly equivalent indicating that the inhibition of transformation largely resulted from inhibition of cell colony formation. Proliferation and transformation were unaffected when [Ca 2+] was reduced to 0.2 mM on days 3–6. Reduction to 0.01 mM Ca 2+ during the same period, however, completely abolished transformation by BP, UV-irradiation or n-acetoxyacetylaminofluorene (AcAAF) without reducing cell proliferation. Thus, the expression of morphological transformation in newly transformed hamster cells is dependent upon extracellular [Ca 2+] to a greater degree than is cell proliferation.

Generation of CFU-C/suppressor T cells in vitro: an experimental model for immune-mediated marrow failure

T cells were derived from the bone marrow of 8 healthy donors and fractionated, according to their receptors for the Fc fragment of IgG, into TG+ and TG- lymphocytes. These were then cocultured with autologous or allogeneic bone marrow cells in agar in the CFU-C assay. No significant suppresion of colony formation could be detected. Total T, TG+, and TG- cells were then incubated for 18 hr with PWM, washed, and cocultured with bone marrow cells. PWM-treated TG- cells showed no significant CFU-C suppressor activity, whereas PWM-treated total T and TG+ cells inhibited colony formation of both autologous and allogeneic marrow cells. The supernatant of PWM-treated total T and TG+ cells also inhibited colony formation. PWM alone enhanced colony formation. The results of this study indicate that normal T cells can be activated in vitro to become CFU-C/suppressor cells after PWM stimulation, and that this effect is mediated by T cells with the Fc receptor for IgG.

Generation of CFU-C/Suppressor T Cells In Vitro: An Experimental Model for Immune-Mediated Marrow Failure

T cells were derived from the bone marrow of 8 healthy donors and fractionated, according to their receptors for the Fc fragment of IgG, into TG+ and TG- lymphocytes. These were then cocultured with autologous or allogeneic bone marrow cells in agar in the CFU-C assay. No significant suppresion of colony formation could be detected. Total T, TG+, and TG- cells were then incubated for 18 hr with PWM, washed, and cocultured with bone marrow cells. PWM-treated TG- cells showed no significant CFU-C suppressor activity, whereas PWM-treated total T and TG+ cells inhibited colony formation of both autologous and allogeneic marrow cells. The supernatant of PWM-treated total T and TG+ cells also inhibited colony formation. PWM alone enhanced colony formation. The results of this study indicate that normal T cells can be activated in vitro to become CFU-C/suppressor cells after PWM stimulation, and that this effect is mediated by T cells with the Fc receptor for IgG.

Granulopoietic studies in acute lymphocytic leukemia of children

Studies have been carried out on the levels of serum and urine colony stimulating activity (CSA) and peripheral blood and bone marrow colony forming cell numbers in children with acute lymphocytic leukemia (ALL) during various phases of their disease. These studies have suggested that serum and urine levels of colony stimulating factor are reduced during the inital or relapse phase of the disease compared to levels found during remission. It has also been found that the number of bone marrow colony forming cells is reduced in relapse or before treatment and elevated during remission while the number of peripheral blood colony forming cells is increased during relapse or before treatment and normal during remission. It has also been shown that mixing of serum or leukemic cells with normal human bone marrow cells inhibits colony formation.