Cell Colony Formation Capacity Research Articles

Mir-186 inhibits the proliferation and growth of multiple myeloma cells by targeting Jagged-1 expression

Purpose: To investigate the suppressive influence of mir-186 on multiplication and growth of multiple myeloma (MM), as well as the processes involved.Methods: The U266 cells were divided into control, mir-186 overexpression, and inhibition groups. The latter two were transfected with mir-186 agent and antagonist, respectively. Cell viability, colony formation potential, cell cycle ratio, and Jagged-1 mRNA and protein levels were measured using various assays.Results: Cell growth increased over time in all groups. However, mir-186 overexpression cells showed significantly decreased growth and colony formation capacity, relative to control, while the mir-186 inhibition cells showed significantly higher growth and colony formation capacity. The study revealed a higher proportion of G0/G1 stage cells and lower proportion of S-phase cells in mir-186 overexpression cells than in control cells. The opposite effect was seen in mir-186 inhibition cells. Jagged-1 protein and mRNA levels were significantly lower in mir-186 overexpression cells and higher in mir-186 inhibition cells than in control group. The Jagged-1 knockout group showed significantly higher Jagged-1 mRNA and protein levels than both the control and mir-186 overexpression groups (p < 0.05).Conclusion: When overexpressed, mir-186 inhibits the growth of multiple myeloma cells by inhibiting cell colony-formation capacity and by regulating cell cycle through mir-186-induced regulation of the expression of Jagged-1. there is need for more research to confirm the clinical benefits of therapies based on these findings.

Tomentosin induces apoptosis in pancreatic cancer cells through increasing reactive oxygen species and decreasing mitochondrial membrane potential

The aim of this study was to determine possible anticancer effect of tomentosin, a natural sesquiterpene lactone, on pancreatic cancer cells. The cytotoxic effect of tomentosin was determined by XTT analysis. Colony formation and apoptosis analyzes were performed, Reactive oxygen species (ROS) level and change in mitochondrial membrane potential (MMP) were evaluated in control and tomentosin-treated cells. The effect of tomentosin on expression levels of apoptosis-related genes was determined by qRT-PCR and Caspase-3 and Caspase-9 proteins were analyzed by western blot. And, the effect of tomentosin on migration and invasion of cells were evaluated. The IC50 dose of tomentosin was found to be 31.11 μM in PANC-1 cells and 33.93 μM in MIA PaCa-2 cells for 48 h. And, treatment of tomentosin at IC50 dose suppressed the colony forming capacity of cells. While tomentosin increased apoptosis rate and ROS production, an decrease was observed in MMP. Tomentosin affected expression level of apoptosis-related genes and increased Caspase-3 and Caspase-9 protein levels. After tomentosin treatment, cell migration and invasion were suppressed. As a result, this study reveals that tomentosin has anticancer effects on pancreatic cancer cells, and therefore it predicts that tomentosin can be evaluated as an effective agent against pancreatic cancer.

Effect of cucurbitacin on malignant biological behavior of breast cancer cells, and its possible underlying mechanism

Purpose: To study the influence of cucurbitacin on malignant biological behavior of mammary carcinoma cells, and the likely mechanism involved.Methods: Human mammary carcinoma cell line MDA-MB-436 was selected for cell culture and treated with different concentrations of cucurbitacin. The effect of cucurbitacin on cell activity, cell colonyformation capacity, cell invasion, migration potential, matrix metalloproteinase-9 (MMP-9) activity, and levels of vascular endothelial growth factor A (VEGFA), epithelial calcium adhesion (E-cadherin), and neurogenic calcium adhesion (N-cadherin) were measured. Moreover, levels of wave protein (vimentin), phosphorylated epidermal growth factor receptor (p-EGFR), phosphorylated signaling transduction, and transcription activation factor 3 (p-STAT3) and phosphorylated protein kinase B (p-Akt) were determined.Results: With increase in cucurbitacin dose, there was significant decrease in cell viability, cell colony ratio, cell invasion and migration capacity, and expression levels of MMP-9, VEGFA, e-cadherin, ncadherin, vimentin, P-EGFR, P-STAT3 and p-Akt (p < 0.05).Conclusion: Cucurbitacin inhibits the proliferation, invasion, and migration of breast cancer cells by down-regulating the expressions of EGFR/STAT3/Akt signaling-related proteins, and inhibiting epithelial-mesenchymal transition transformation.

A Small Molecule Inhibitor, OGP46, Is Effective against Imatinib-Resistant BCR-ABL Mutations via the BCR-ABL/JAK-STAT Pathway

Chronic myeloid leukemia (CML) is caused by the Philadelphia (Ph+) chromosome carrying the BCR-ABL oncogene, a constitutively active tyrosine kinase. The discovery of imatinib represents a major success story in the treatment against CML. However, mutations in the BCR-ABL kinase domain are a major cause of resistance to imatinib, demonstrating that BCR-ABL remains a critical drug target. Here, we investigate a novel small molecule inhibitor, OGP46, for its inhibitory activity against K562, a panel of murine BaF3 cell lines stably expressing either wild-type BCR-ABL or its mutant forms, including T315I. OGP46 exhibits potent activity against imatinib-resistant BCR-ABL mutations, including T315I. OGP46 induced cell differentiation accompanied by G0/G1 cell-cycle arrest and suppressed the colony formation capacity of cells. Treatment with OGP46 significantly decreased the mRNA and protein expression of BCR-ABL in K562 and BaF3-p210-T315I cells. Mechanistically, the anti-cancer activity of OGP46 induced by cell differentiation is likely through the BCR-ABL/JAK-STAT pathway in native BCR-ABL and mutant BCR-ABL, including T315I, of CML cells. Our findings highlight that OGP46 is active against not only native BCR-ABL but also 11 clinically relevant BCR-ABL mutations, including T315I mutation, which are resistant to imatinib. Thus, OGP46 may be a novel strategy for overcoming imatinib-resistance BCR-ABL mutations, including T315I.

LINC00473 promotes hepatocellular carcinoma progression via acting as a ceRNA for microRNA-195 and increasing HMGA2 expression

Hepatocellular carcinoma (HCC) is a most aggressive malignant tumor. Nevertheless, the molecular mechanisms underlying HCC are still completely unclear. LINC00473 is identified as a tumor promoter in many cancers. In this investigation, the function of LINC00473 was specifically focused on. We exhibited that LINC00473 was obviously elevated in HCC cells compared to QSG-7701 cells. Functionally, down-regulation of LINC00473 could prevent HCC cell viability and cell proliferation. For another, HCC cell colony formation capacity was greatly restrained while cell apoptosis was triggered by loss of LINC00473. Meanwhile, would-healing assay and transwell invasion experiments were employed in our present study. As demonstrated, we observed that HCC cell migratory and invasive ability were obviously suppressed by the silence of LINC00473. Apart from these, mechanistic investigations implied miR-195 was a sponge target of LINC00473. It is widely established miR-195 is a famous tumor inhibitory gene regulator in various cancers. Here, we confirmed the binding correlation between LINC00473 and miR-195 using RIP assay. Subsequently, in vivo experiments were employed and it was manifested that LINC00473 was able to promote HCC tumor growth via acting as a ceRNA to inhibit miR-195. HMGA2 is a kind of nuclear-binding protein and it is involved in various cancers. We predicted it as a target of miR-195 and we confirmed their correlation. In addition, HMGA2 was repressed by loss of LINC00473, which was rescued by miR-195 inhibitors. Then, we found that angiogenic fator vascular endothelial growth factor (VEGF) was inhibited by loss of LINC00473 whereas anti-angiogenic factor EPN2 was induced in vivo. Taken all these together, our study revealed the significance of LINC00473/miR-195/HMGA2 signaling axis for the first time in HCC progression. It was suggested the potential possibility of LINC00473 as an indicator for HCC.

Abstract 2611: Activated pentose phosphate pathway mediated by FBP1 upregulation supports progression of acute myeloid leukemia with high EVI1 expression

Abstract EVI1 (ecotropic viral integration site 1) is a transcription factor essential for the development and progression of acute myeloid leukemia (AML). Since high EVI1 expression, seen in approximately 10% of AML patients (EVI1high AML), is associated with poor clinical outcome, development of a novel therapeutic strategy is required. In this study, we investigated key molecules that are induced by aberrant EVI1 expression and modulate the metabolomics of leukemia cells. We conducted whole transcriptome analysis by RNA-sequencing in murine leukemia cells induced by retroviral transduction with EVI1 followed by transplantation into irradiated mice. We identified 45 genes with more than 10-fold expression in EVI1-transduced bone marrow cells compared to those with the mock vector at both pre-leukemic phase and leukemia phase. Intriguingly, the fructose bisphosphatase 1 gene (Fbp1), which encodes a key enzyme of gluconeogenesis was highly upregulated in EVI1-induced AML cells. We confirmed that FBP1 mRNA expression is quickly upregulated by ectopic expression of EVI1 in murine LSK (Linneg, c-Kitpos, Sca-1pos) cells. Moreover, we observed an enrichment of EVI1 in the promoter and enhancer region of Fbp1 by chromatin immunoprecipitation followed by qPCR analysis in murine hematopoietic cells, suggesting that Fbp1 expression is directly regulated by EVI1. Although high FBP1 expression suppresses glycolytic metabolism and negatively affects cellular proliferation in various types of solid tumor, its role in AML has not been investigated. In contrast to the results previously reported in other types of cancers, pharmacologic inhibition or short hairpin RNA (shRNA)-mediated knockdown of FBP1 significantly decreased colony-forming cell (CFC) capacity in EVI1-transduced murine LSK cells and significantly delayed progression of leukemia in the mice secondarily transplanted with EVI1-induced AML cells in vivo. These results suggest that aberrant expression of FBP1 is important for progression of EVI1high AML. While high FBP1 enzymatic activity negatively affects the glycolytic pathway, it in turn enhances the pentose phosphate pathway (PPP) through elevating the fructose-6-phosphate level. Activated PPP results in enhanced nucleotide synthesis essential for rapidly dividing cells. Importantly, individual knockdown of main PPP enzymes (G6pd, Pgd, Rpia) by shRNA transduction significantly decreased the CFC capacity of EVI1 transduced murine LSK cells, further confirming an important role of PPP in driving proliferation of EVI1high leukemia cells. Collectively, these results indicate that the activated PPP through transcriptional upregulation of FBP1 is crucial for progression of EVI1-driven leukemia. Since inhibition of FBP-1 did not compromise normal hematopoiesis, targeting the enzyme can be a promising therapeutic approach for the EVI1high AML. Citation Format: Hideaki Mizuno, Yuki Kagoya, Akira Chiba, Junji Koya, Yosuke Masamoto, Mineo Kurokawa. Activated pentose phosphate pathway mediated by FBP1 upregulation supports progression of acute myeloid leukemia with high EVI1 expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2611.

PTPN2 induced by inflammatory response and oxidative stress contributed to glioma progression.

Malignant glioma remains the most frequent form of primary brain tumors all over the world. The gliomagenesis is characterized by various molecular processes such as neoplastic transformation, dysregulation of the cell cycle, and angiogenesis. Among these biomolecular events, the existence of inflammation and oxidative stress pathways in the development of glioma has been reported. PTPN2 is associated with several inflammatory disorders. However, the biological role of PTPN2 in inflammation responses and oxidative stress pathways involved in glioma remains poorly known. Here, we focused on its function in glioma development. Here, we observed that PTPN2 was significantly increased in glioma especially in a grade-dependent manner. Meanwhile, interferon-γ and tumor necrosis factor-α, which have been identified as crucial inflammation cytokines, were able to trigger PTPN2 expression in a dose-dependent course in T98G cells. Then, we found that PTPN2 was oxidated and inactivated by H2 O2 . Meanwhile, H2 O2 induced glioma cell colony formation capacity and increased ki-67 expression confirmed by flow cytometry assay. Finally, T98G cells were transfected with PTPN2 shRNA and it was shown that knockdown of PTPN2 obviously inhibited T98G cell colony formation and induced cell apoptosis. In summary, our findings indicated that PTPN2 could be induced by inflammatory response and oxidative stress and its deficiency depressed glioma cell growth.

Downregulation of lysyl oxidase-like 4 LOXL4 by miR-135a-5p promotes lung cancer progression in vitro and in vivo.

Aberrant microRNAs are widely identified in multiple cancers, including lung cancer. miR-135a-5p can function as a significant tumor regulator in diverse cancers via impacting multiple genes in oncogenic pathways. Nevertheless, the biological role of miR-135a-5p in lung cancer is poorly known. Here, we investigated its function in lung cancer. As exhibited, miR-135a-5p was elevated in lung cancer cells in contrast to BEAS-2B cells. Then, we inhibited miR-135a-5p expression by transfecting LV-anti-miR-135a-5p into lung cancer cells. As displayed, miR-135a-5p was obviously reduced in A549 and H1299 cells. Knockdown of miR-135a-5p repressed lung cancer cell growth and cell proliferation. Meanwhile, cell colony formation capacity was depressed, cell apoptosis was enhanced and cell cycle progression was blocked in G1 phase by inhibition of miR-135a-5p in vitro. Additionally, the migration and invasion of A549 and H1299 cells was strongly depressed by LV-anti-miR-135a-5p. For another, by using informatics analysis, lysyl oxidase-like 4 (LOXL4) was speculated as the downstream target of miR-135a-5p. We validated their direct correlation and moreover, overexpression of miR-135a-5p restrained LOXL4 levels in lung cancer cells. Subsequently, we proved that miR-135a-5p promoted lung cancer development via targeting LOXL4 by carrying out the in vivo assays. Taken these together, our study revealed miR-135a-5p might be indicated as a perspective for lung cancer via targeting LOXL4.

Silencing non-SMC chromosome-associated polypeptide G inhibits proliferation and induces apoptosis in hepatocellular carcinoma cells.

The present study was designed to investigate the significance of non-structural maintenance of chromosomes (non-SMC) chromosome-associated polypeptide G (NCAPG), a subunit of condensin complex I, in the development of hepatocellular carcinoma (HCC). NCAPG protein expression in human HCC and paracancerous hepatic tissues were examined using immunohistochemistry, and NCAPG mRNA expression in HCC cell lines were quantified using quantitative RT-PCR. Lentivirus-mediated RNA interference was used to silence NCAPG in HCC cells. Cell proliferation was monitored by MTT assay. Cell colony-forming capacity was measured by colony formation assay. Apoptosis was determined by flow cytometry. The results showed that increased protein expression of NCAPG was found in HCC tissues compared with the matched paracancerous hepatic tissues. At the mRNA level, increased expression of NCAPG was found in HCC cells as opposed to the normal hepatocytes. Silencing of NCAPG in BEL-7404 and SMMC-7721 cells led to decreased cell proliferation and increased apoptosis. These changes were associated with increased mRNA expressions of P53, P27, and Bad, but decreased mRNA expression of EGFR, Akt, survivin, and JNK. NCAPG might play an oncogenic role in the development of liver cancer. Further studies to clarify its role and underlying mechanisms in the development of liver cancer are warranted.

LncRNA BX357664 inhibits cell proliferation and invasion and promotes cell apoptosis in human colorectal cancer cells.

Colorectal cancer represents a great burden for patients worldwide. Long noncoding RNA BX357664 is an RNA that was identified by microarray technique in renal cell carcinoma. The function of BX357664 in solid tumors remains largely unknown. The present study aimed to investigate the expression profile and functional role of BX357664 in human colorectal cancer progression. The transcription levels of BX357664 were initially examined in vivo and in vitro. An overexpression plasmid was constructed in order to examine the effects of BX357664 overexpression on cell proliferation, apoptosis, migration and invasion. The results demonstrated that BX357664 was significantly downregulated in clinical colorectal cancer tissues and cell lines. Overexpression of BX357664 decreased cell proliferation rates and cell colony formation capacities in HCT116 and HT-29 cells. Following BX357664 overexpression, HCT116 and HT-29 cells exhibited reduced migration and invasion capacities. Would closure was also blunted by >50% following overexpression of BX357664 in HCT-116 and HT-29 cells. In addition, the cell cycle regulators Cyclin B1, CDC25C and Cyclin D1 as well as the mesenchymal marker N-cadherin were downregulated, whereas the epithelial marker E-cadherin was upregulated by BX357664 overexpression. Finally, HCT116 and HT-29 cell apoptosis was induced and activities of caspase-3 and caspase-9 increased significantly following BX357664 overexpression. The present data suggested that BX257664 negatively regulated cell proliferation and metastasis and promoted cell apoptosis in colorectal cancer. These observations provided novel evidence that BX357664 might serve as a tumor suppressor and a potential therapeutic target in the treatment of colorectal cancer in the clinic.

Loss of ASXL1 in the bone marrow niche dysregulates hematopoietic stem and progenitor cell fates

Somatic or de novo mutations of Additional sex combs-like 1 (ASXL1) frequently occur in patients with myeloid malignancies or Bohring-Opitz syndrome, respectively. We have reported that global loss of Asxl1 leads to the development of myeloid malignancies and impairs bone marrow stromal cell (BMSC) fates in mice. However, the impact of Asxl1 deletion in the BM niche on hematopoiesis remains unclear. Here, we showed that BMSCs derived from chronic myelomonocytic leukemia patients had reduced expression of ASXL1, which impaired the maintaining cord blood CD34+ cell colony-forming capacity with a myeloid differentiation bias. Furthermore, Asxl1 deletion in the mouse BMSCs altered hematopoietic stem and progenitor cell (HSC/HPC) pool and a preferential myeloid lineage increment. Immunoprecipitation and ChIP-seq analyses demonstrated a novel interaction of ASXL1 with the core subunits of RNA polymerase II (RNAPII) complex. Convergent analyses of RNA-seq and ChIP-seq data revealed that loss of Asxl1 deregulated RNAPII transcriptional function and altered the expression of genes critical for HSC/HPC maintenance, such as Vcam1. Altogether, our study provides a mechanistic insight into the function of ASXL1 in the niche to maintain normal hematopoiesis; and ASXL1 alteration in, at least, a subset of the niche cells induces myeloid differentiation bias, thus, contributes the progression of myeloid malignancies.

Effect of FAM196B in human lung adenocarcinoma.

Lung cancer is the leading cause of cancer-related mortality worldwide. The present study focused on the function of family with sequence similarity 196 member B (FAM196B) in lung adenocarcinoma (LUAD). We analyzed lung carcinoma patients' data from the Cancer Genome Atlas (TCGA), and verify the change of FAM196B expression in 30 LUAD tissues and matched adjacent non-tumor tissues (> 5 cm) by tissue microarray. To investigate the role of FAM196B in LUAD, we used lentivirus-mediated short hairpin RNA (shRNA) to knockdown FAM196B expression in human LUAD cell line A549 and H1299 and assessed it by RT-qPCR and western blot. Celigo Imaging Cytometry System, MTT assays and colony formation were conducted to evaluate cell proliferation. Cell apoptosis were assayed by Annexin V staining. We found that FAM196B was significantly upregulated (P=5.06E-06) in LUAD compared with adjacent non-tumor tissues. Cell proliferation was inhibited in FAM196B-silenced A549 and H1299 cells using Celigo Imaging Cytometry System, MTT assays and colony formation assays. Apoptosis rate was significantly increased in FAM196B-shRNA group than the control group. In conclusion, Knockdown of FAM196B can inhibit cell proliferation, cell colony formation capacity, and promote cell apoptosis in A549 and H1299 cell lines. FAM196B may be one novel potential targets for treating patients with LUAD.

Knockdown of DDX46 inhibits proliferation and induces apoptosis in esophageal squamous cell carcinoma cells.

Esophageal squamous cell carcinoma (ESCC) is the most common type of esophageal carcinoma and remains the leading cause of cancer-related death worldwide. DEAD-box RNA helicases play critical roles in cellular metabolism and in many cases have been implicated in cellular proliferation and neoplastic transformation. DDX46 belongs to DEAD-box helicase family, the expression pattern of DDX46 in ESCC tissues and the biologic role in ESCC progression have not been implicated previously. In this study, DDX46 expression in human ESCC and adjacent normal tissues were explored using immunohistochemistry, and ESCC cell lines compared with normal esophageal epithelium cell were quantified using real‑time PCR. Next, lentivirus-mediated RNA interference was applied to silence DDX46 in TE-1 and Eca-109 cells. Cell growth was monitored using high content screening. Cell viability was measured by MTT assay. Cell colony-forming capacity was measured by colony formation assay. Cell cycle progression and apoptosis were determined by flow cytometry. Further, the stress and apoptosis signaling antibody array kit was used to detect the changes of signaling molecules in TE-1 cells after DDX46 knockdown. We found that DDX46 was significantly upregulated in ESCC tissues and cells compared with normal tissues and cells. DDX46 knockdown led to decreased proliferation and increased apoptosis in TE-1 and Eca-109 cells. Moreover, DDX46 silencing resulted in apoptotic induction via decreased phosphorylation of Akt and IκBα, as well as negative regulation of NF-κB signaling. In conclusion, these results demonstrate that DDX46 knockdown inhibited cell growth, and induced apoptosis, suggest that DDX46 is critical for ESCC cells proliferation. In addition, this study provides a foundation for further study into the clinical potential diagnosis and novel therapeutic target for ESCC.

Pretreatment with the γ-secretase inhibitor DAPT sensitizes drug-resistant ovarian cancer cells to cisplatin by downregulation of Notch signaling

Notch signaling is implicated in ovarian cancer tumorigenesis and inhibition of Notch signaling with γ-secretase inhibitor DAPT resulted in reduction of tumor cell viability and induction of apoptosis in ovarian cancer cells. This study investigated whether DAPT has the same effect on ovarian cancer cells that are resistant to cisplatin and the underlying molecular events. Ovarian cancer cell lines resistant to cisplatin were treated with DAPT, cisplatin or combination for cell viability MTT, flow cytometric cell cycle, ELISA apoptosis and colony formation assays. qRT-PCR and western blotting were used to detect gene expressions. We found that pretreatment of ovarian cancer cisplatin-resistant cell lines with DAPT for 24 h and then with cisplatin for 72 h showed a synergistic antitumor activity in these cell lines, while cisplatin treatment and then addition of DAPT just showed an additive or antagonistic effects on these cisplatin-resistant ovarian cancer cells. Moreover, pretreatment of ovarian cancer cell lines with DAPT and then with cisplatin also inhibited tumor cell colony formation capacity, arrested tumor cells at G2 phase of the cell cycle and induced apoptosis. The cell cycle and apoptosis-related genes, such as cyclin B1, Bcl-2 and caspase-3, were also modulated by the treatment. Pretreatment of ovarian cancer cell lines with DAPT and then with cisplatin downregulated Notch1 and Hes1 expression dose- and time-dependently. The current data demonstrate that DAPT pretreatment was able to sensitize cisplatin-resistant human ovarian cancer cells to cisplatin by downregulation of Notch signaling.

Micronucleus formation induced by dielectric barrier discharge plasma exposure in brain cancer cells

Induction of micronucleus formation (cytogenetic damage) in brain cancer cells upon exposure of dielectric barrier discharge plasma has been investigated. We have investigated the influence of exposure and incubation times on T98G brain cancer cells by using growth kinetic, clonogenic, and micronucleus formation assay. We found that micronucleus formation rate directly depends on the plasma exposure time. It is also shown that colony formation capacity of cells has been inhibited by the treatment of plasma at all doses. Cell death and micronucleus formation are shown to be significantly elevated by 120 and 240 s exposure of dielectric barrier discharge plasma.

Involvement of CCR6/CCL20/IL-17 Axis in NSCLC Disease Progression

ObjectivesAutocrine and paracrine chemokine/chemokine receptor-based interactions promote non-small-cell-lung-cancer (NSCLC) carcinogenesis. CCL20/CCR6 interactions are involved in prostatic and colonic malignancy pathogenesis. The expression and function of CCL20/CCR6 and its related Th-17 type immune response in NSCLC is not yet defined. We sought to characterize the role of the CCL20/CCR6/IL-17 axis in NSCLC tumor growth.MethodsA specialized histopathologist blindly assessed CCL20/CCR6 expression levels in 49 tissue samples of NSCLC patients operated in our department. Results were correlated to disease progression. Colony assays, ERK signaling and chemokine production were measured to assess cancer cell responsiveness to CCL20 and IL-17 stimulation.ResultsCCL20 was highly expressed in the majority (38/49, 77.5%) of tumor samples. Only a minority of samples (8/49, 16.5%) showed high CCR6 expression. High CCR6 expression was associated with a shorter disease-free survival (P = 0.008) and conferred a disease stage-independent 4.87-fold increased risk for disease recurrence (P = 0.0076, CI 95% 1.52–15.563). Cancerous cell colony-forming capacity was increased by CCL20 stimulation; this effect was dependent in part on ERK phosphorylation and signaling. IL-17 expression was detected in NSCLC; IL-17 potentiated the production of CCL20 by cancerous cells.ConclusionOur findings suggest that the CCL20/CCR6 axis promotes NSCLC disease progression. CCR6 is identified as a potential new prognostic marker and the CCL20/CCR6/IL-17 axis as a potential new therapeutic target. Larger scale studies are required to consolidate these observations.

Gender Differences in Circulating Endothelial Progenitor Cell Colony-Forming Capacity and Migratory Activity in Middle-Aged Adults

Middle-aged women have a lower prevalence and incidence of cardiovascular events compared with men. The mechanisms responsible for this gender-specific difference are unclear. Numeric and functional impairments of bone marrow-derived circulating endothelial progenitor cells (EPCs) are associated with increased cardiovascular and cerebrovascular morbidity and mortality. It is currently unknown whether there are gender-related differences in EPC number and function in middle-aged adults. We tested the hypothesis that EPCs isolated from middle-aged women demonstrate greater colony-forming capacity and migratory activity compared with men of similar age. Peripheral blood samples were collected from 50 sedentary adults, 25 men (59 +/- 1 years of age) and 25 women (58 +/- 1 years of age). Mononuclear cells were isolated and preplated for 2 days, and nonadherent cells were further cultured for 7 days to determine EPC colony-forming units. Migratory activity of EPCs was determined using a modified Boyden chamber. The number of EPC colony-forming units was significantly higher (approximately 150%) in samples collected from women (16 +/- 3) compared with that collected from men (7 +/- 1). In addition, EPC migration (relative fluorescent units) was approximately 40% greater in women (729 +/- 74) than in men (530 +/- 67). In conclusion, these results demonstrate that EPC colony-forming capacity and migratory activity are higher in middle-aged women than in men.

Oligonucleotide Enhanced Cytotoxicity of Idarubicin for Lymphoma Cells

Oligonucleotides offer the potential to manipulate gene expression in targeted cells which might be exploitable for therapeutic benefit. The effects of combining a phosphorothioate oligonucleotide OL(1) p53, which transiently down-regulates p53 levels, with an anthracycline, Idarubicin, on the growth of wild-type p53 WMN gene-expressing lymphoma cells was evaluated. Fluorescent OL(1) p53, was used to demonstrate oligonucleotide uptake and retention by the WMN cells. Uptake was maximal at 24 hours and compared to baseline (0 hours) increasing apoptotic cells were evident in WMN cells treated with OL(1) (1 μM) alone and in combination with Idarubicin (0.2 nM) for 24 to 48 hours. In cells treated with OL(1) p53 and Idarubicin, truncated p53 message of a predicted 201 base pair length based on RNAase H cleavage of the OL(1) p53-p53 mRNA heteroduplex was detected after 7 hours of incubation. The message for p53 was transiently downregulated as detected by RT-PCR analysis at 24 hours, and protein levels transiently reduced at 36 hours, as shown by a quantitative Western blot. Corresponding to these events, the growth of WMN cells ceased after 48 hours in the concurrent presence of OL(1) p53 and Idarubicin and, the lymphoma cells were dead after 72 hours. No reduction in hematopoietic colony forming cell capacity of similarly treated hematopoietic progenitor cells harvested from cytokine-mobilized blood by apheresis was observed. Therefore, synergistic cytotoxicity of Idarubicin for lymphoma cells treated with an oligonucleotide targeting p53 message was demonstrated at oligonucleotide and Idarubicin concentrations which were minimally toxic to hematopoietic progenitor cells. This approach offers new opportunities for purging of lymphoma cells from hematopoietic harvests and systemic lymphoma therapy.

Impaired in vitro growth of PHA induced T lymphocyte colonies in hemodialyzed renal failure patients

Using an in vitro method that allows the study of the colony forming capacity of phytohemagglutinin stimulated peripheral blood T lymphocytes, we have detected an impaired T cell colony formation in hemodialyzed renal failure patients. By contrast a near normal pattern of responses was observed in patients treated with a conservative therapy. The poor in vitro T cell responsiveness of hemodialyzed patients was not corrected by supplementing the cultures with an adherent cell contitioned medium prepared from normal donors. We conclude that an intrinsic defect of the T cell colony forming capacity exists in hemodialyzed patients.

Quantitative Bestimmung der Zellwachstumshemmung

The influence of five cytostatic substances on the colony-forming capacity of cells of the strain Detroit 6 was examined, and the advantages of this method for thein vitro screening of cytostatic substances was pointed out.