MicroRNA-222 contributed to cell proliferation, invasion and migration via regulating YWHAG in osteosarcoma.

OBJECTIVE: The aim of this study was to investigate the role of microRNA-222 (miR-222) in osteosarcoma (OS), and to further explore the potential molecular mechanism. PATIENTS AND METHODS: We measured the level of miR-222 in OS tissues and cell lines using quantitative Real-time polymerase chain reaction. Synthesized miR-222 mimics or inhibitors were obtained to up-regulate or down-regulate the expression of miR-222 in U2OS or Saos2 cells. Cell counting kit-8 (CCK8) and colony formation assay were employed to detect the ability of cell proliferation, and transwell assay was used to confirm the ability of cell invasion. Furthermore, luciferase assay and Western blot were applied to verify the target of miR-222 in OS. RESULTS: The level of miR-222 in OS tumor tissue samples was significantly lower than that in normal group. Over-expression of miR-222 decreased cell proliferation and invasion in U2OS cells while knockdown of miR-222 promoted cell growth and metastasis in Saos2 cells. Furthermore, YWHAG was found to be a candidate target of miR-222 using several databases. Elevated level of miR-222 inhibited YWHAG expression while reduced miR-222 promoted YWHAG expression. Also, up-regulation of YWHAG restored the inhibiting effect of miR-222 mimics. CONCLUSIONS: We identified for the first time that the expression level of miR-222 was reduced in OS tissues as well as in OS cell lines. miR-222 could inhibit cell proliferation and invasion via down-regulating YWHAG. These data could provide a potential target for the biological treatment of OS.

Time-to-Event Analysis of Polatuzumab Vedotin-Induced Peripheral Neuropathy to Assist in the Comparison of Clinical Dosing Regimens.

Polatuzumab vedotin, an antibody-drug conjugate containing monomethyl auristatin E, was associated with an incidence of grade ≥2 peripheral neuropathy (PN) of 55-72% in patients with indolent non-Hodgkin lymphoma in a phase II study, when dosed 1.8-2.4 mg/kg every 3 weeks until progression or for a maximum of 17 cycles. To quantify the correlation of conjugate exposure and treatment duration with PN risk, a time-to-event model was developed using data from phase I and II studies. The model suggested that PN risk increased with conjugate exposure and treatment cycles, and a trend for increased risk with body weight and albumin concentration. When capping the treatment duration to six to eight cycles, the risk ratio of a dose of 2.4 mg/kg vs. 1.8 mg/kg was ≥1.29; the predicted incidence of grade ≥2 PN at 1.8-2.4 mg/kg dose levels was 17.8-37.2%, which is comparable with other antimicrotubule agents for lymphoma treatment.

Integrated Two-Analyte Population Pharmacokinetic Model for Antibody-Drug Conjugates in Patients: Implications for Reducing Pharmacokinetic Sampling.

An integrated pharmacokinetics (PK) model that simultaneously describes concentrations of total antibody (Tab) and antibody-conjugated monomethyl auristatin E (acMMAE) following administration of monomethyl auristatin E (MMAE)-containing antibody-drug conjugates (ADCs) was developed based on phase I PK data with extensive sampling for two ADCs. Two linear two-compartment models that shared all parameters were used to describe the PK of Tab and acMMAE, except that the deconjugation rate was an additional clearance pathway included in the acMMAE PK model compared to Tab. Further, the model demonstrated its ability to predict Tab concentrations and PK parameters based on observed acMMAE PK and various reduced or eliminated Tab PK sampling schemes of phase II data. Thus, this integrated model allows for the reduction of Tab PK sampling in late-phase clinical development without compromising Tab PK characterization.

Anti-CD22 and anti-CD79B antibody drug conjugates are active in different molecular diffuse large B-cell lymphoma subtypes.

Antibody drug conjugates (ADCs), in which cytotoxic drugs are linked to antibodies targeting antigens on tumor cells, represent promising novel agents for the treatment of malignant lymphomas. Pinatuzumab vedotin is an anti-CD22 ADC and polatuzumab vedotin an anti-CD79B ADC that are both linked to the microtubule-disrupting agent monomethyl auristatin E (MMAE). In the present study, we analyzed the activity of these agents in different molecular subtypes of diffuse large B-cell lymphoma (DLBCL) both in vitro and in early clinical trials. Both anti-CD22-MMAE and anti-CD79B-MMAE were highly active and induced cell death in the vast majority of activated B-cell-like (ABC) and germinal center B-cell-like (GCB) DLBCL cell lines. Similarly, both agents induced cytotoxicity in models with and without mutations in the signaling molecule CD79B. In line with these observations, relapsed and refractory DLBCL patients of both subtypes responded to these agents. Importantly, a strong correlation between CD22 and CD79B expression in vitro and in vivo was not detectable, indicating that patients should not be excluded from anti-CD22-MMAE or anti-CD79B-MMAE treatment because of low target expression. In summary, these studies suggest that pinatuzumab vedotin and polatuzumab vedotin are active agents for the treatment of patients with different subtypes of DLBCL.

Suppression of tumor angiogenesis by targeting the protein neddylation pathway.

Inhibition of protein neddylation, particularly cullin neddylation, has emerged as a promising anticancer strategy, as evidenced by the antitumor activity in preclinical studies of the Nedd8-activating enzyme (NAE) inhibitor MLN4924. This small molecule can block the protein neddylation pathway and is now in clinical trials. We and others have previously shown that the antitumor activity of MLN4924 is mediated by its ability to induce apoptosis, autophagy and senescence in a cell context-dependent manner. However, whether MLN4924 has any effect on tumor angiogenesis remains unexplored. Here we report that MLN4924 inhibits angiogenesis in various in vitro and in vivo models, leading to the suppression of tumor growth and metastasis in highly malignant pancreatic cancer, indicating that blockage of angiogenesis is yet another mechanism contributing to its antitumor activity. At the molecular level, MLN4924 inhibits Cullin-RING E3 ligases (CRLs) by cullin deneddylation, causing accumulation of RhoA at an early stage to impair angiogenic activity of vascular endothelial cells and subsequently DNA damage response, cell cycle arrest and apoptosis due to accumulation of other tumor-suppressive substrates of CRLs. Furthermore, we showed that inactivation of CRLs, via small interfering RNA (siRNA) silencing of its essential subunit ROC1/RBX1, recapitulates the antiangiogenic effect of MLN4924. Taken together, our study demonstrates a previously unrecognized role of neddylation in the regulation of tumor angiogenesis using both pharmaceutical and genetic approaches, and provides proof of concept evidence for future development of neddylation inhibitors (such as MLN4924) as a novel class of antiangiogenic agents.

CD4+Foxp3+ regulatory T-cell impairment by paclitaxel is independent of toll-like receptor 4.

Paclitaxel (PTX) is one of the most widely used clinical antitumour drugs in chemotherapy nowadays. Its effect on immune system has become a hot spot of research in recent years. Here, we demonstrated that PTX not only decreased the percentage of CD4+Foxp3+ regulatory T (Treg) cells both in vitro and in vivo but also impaired cell viability and cytokine production of Treg cells rather than CD4+Foxp3- effector T (Teff) cells. As PTX has been reported to mimic the activity of LPS to trigger the toll-like receptor 4 (TLR4) signalling pathway in macrophages, we investigated the possible role of TLR4 in the effect of PTX. However, although TLR4 expression on Treg cells was higher than that on Teff cells, the expression level remained unaltered in both Treg and Teff cells after PTX treatment. Surface molecules and activation markers in Treg and Teff cells did not change, either. Further study showed that the effect of PTX on TLR4-/- mice deficient in TLR4 signalling was similar to that on C57BL/6 mice both in vivo and in vitro. These data indicate that the selective impairment of Treg cells by PTX is independent of TLR4.

Insulin-like growth factor binding protein-3 (IGFBP-3) acts as an invasion-metastasis suppressor in ovarian endometrioid carcinoma.

Metastasis and invasion occur in the majority of epithelial ovarian carcinoma at diagnosis. To delineate the molecular signature in ovarian cancer invasion, we established and characterized a human ovarian endometrioid carcinoma (EC) cell line OVTW59-P0 and its invasion-related sublines (P1-P4, in the order of increasing invasive activity). P4 showed faster migration and larger xenograft formation with metastasis than P0. By microarray analysis of different gene expression among P0-P4 sublines, one group of gene was found negatively correlated with cancer invasion. Among these genes, IGFBP-3 was identified as one of the most remarkably suppressed gene that showed lower gene expression in P4 than P0. Re-expression of IGFBP-3 in P4 effectively inhibited cell migration, invasion and metastasis, but did not affect cell proliferation. In 35 patients with EC tumors, low IGFBP-3 expression correlated clinically with higher tumor grade, advanced stage and poor survival. Our results provide evidence and indicate that IGFBP-3 plays an important role as an invasion-metastasis suppressor in ovarian EC.

IMP-4, a novel metallo-beta-lactamase from nosocomial Acinetobacter spp. collected in Hong Kong between 1994 and 1998.

Between 1994 and 1998, 97 imipenem-resistant Acinetobacter isolates were identified at the Prince of Wales Hospital, Hong Kong, China. A bla(IMP) PCR product was obtained from 23 of 35 viable cultures; 12 isolates belonged to genomic DNA group 3, 8 belonged to group 2 (Acinetobacter baumannii), 2 belonged to group 13TU, and 1 belonged to group 1. The bla(IMP) homologues were sequenced from two isolates from genomic DNA group 2 and one isolate each from groups 3 and 13TU. The four sequences included an identical 738-bp open reading frame, predicted to encode a polypeptide of 246 amino acids, with 95.6% homology to IMP-1 and 89.3% homology to IMP-2. The new enzyme, designated IMP-4, was partially purified. It had a pI of 8.0 and was strongly active against imipenem and meropenem, with V(max) values 53 and 8% of that for penicillin G, respectively. Strong activity was also seen against oxyimino-aminothiazolyl cephalosporins but not against aztreonam. Hydrolytic activity was inhibited by EDTA but not by clavulanate or tazobactam. Carbapenem MICs for most bla(IMP)-positive isolates were 4 to 32 microg/ml, but one isolate with the intact gene was susceptible, with imipenem and meropenem MICs of 0.25 and 0.5 microg/ml, respectively. The latter isolate did not produce the band with a pI of 8.0, and gene expression was inferred to have been lost. None of the isolates studied in detail contained extrachromosomal DNA, and carbapenem resistance was not transmissible to Escherichia coli. Nevertheless, the presence of bla(IMP-4) in different genomic DNA groups implies horizontal transfer, and sequences resembling a GTTRRRY integrase-dependent recombination motif were identified in the flanking regions of bla(IMP-4).

Epidemiology and infection control implications of Acinetobacter spp. in Hong Kong.

In a previous study, we showed that Acinetobacter genomic DNA group 3 was the most common species among blood culture isolates and was commonly found on superficial carriage sites of the healthy and the sick, which are different findings from those reported in Europe and North America. We used amplified ribosomal DNA restriction analysis and pulsed-field gel electrophoresis to study further the molecular epidemiology of acinetobacters in our region. Over a study period of 6 weeks with 136 consecutive routine clinical isolates (1.33% of all specimens), genomic DNA groups 2 (Acinetobacter baumannii), 3, and 13TU were obtained from 59 of 69 positive patients. There is a significant difference in the specimen sources of the three genomic DNA groups, with group 13TU being significantly associated with the respiratory tract (chi-square exact test, P = 0.0064). Settle plates showed a significantly heavier environmental load from the intensive care unit (ICU) than from the four surgical wards examined (22 of 70 versus 76 of 120 plates with <5 colonies; chi-square test, P < 0. 0001). Genomic group 3 accounted for 6 of 12 clusters of possibly related strains among patients, between patients and the ICU environment, and in the ICU environment. Genomic groups 2 and 3 accounted for 21% of the 132 genomically identified isolates recovered from 21 of 41 local vegetables, 53 of 74 fish and meat samples, and 22 of 60 soil samples. Group 13TU was present only in patients' immediate surroundings. The role played by the environment and by human carriage should be evaluated in order to devise a cost-effective infection control program pertinent to our situation of acinetobacter endemicity.

Analysis with flow cytometry of green fluorescent protein expression in leukemic cells.

BACKGROUND: The measurement of DNA content with propidium iodide (PI) in cells transfected with expression vectors encoding the green fluorescent protein (GFP) is a useful tool in studying a variety of biological functions of proteins within cells. The purpose of this study was to determine conditions of formaldehyde fixation that permit intracellular GFP fluorescence and adequate DNA histograms to be generated following transient transfection of cells with a GFP-encoding plasmid. Cell cycle analysis was also performed in GFP-positive cells. METHODS: The murine myeloid leukemic cell line, 32Dcl3, was used as the model system. Cells were transfected with a GFP-encoding plasmid (pEGFPC1). Following fixation in different formaldehyde concentrations and permeabilization with 70% ethanol, cells were stained with PI and analyzed by flow cytometry for GFP fluorescence and DNA content. Transfected cells were also analyzed for GFP fluorescence and DNA content following release from nocodazole block. RESULTS: Fixing cells in 0.51-1.75% formaldehyde concentrations prior to ethanol permeabilization resulted in 14-19% of transfected cells being GFP-positive, with acceptable coefficients of variation on the G(1) peak of DNA histograms. Analysis of cells synchronized to and released from the G(2)-M phase by nocodazole suggested that GFP-positive cells, when compared to GFP-negative cells, did not appear to progress out of G(2)-M following release from nocodazole block. Simultaneous detection of GFP fluorescence and DNA content by PI staining is possible following transient transfection of cells with a single expression vector encoding GFP. Our results demonstrate that GFP expression can be detected, using flow cytometry to perform cell cycle analysis in murine leukemic cells.

Profiling expression patterns and isolating differentially expressed genes by cDNA microarray system with colorimetry detection.

A high-density cDNA microarray with colorimetry detection system to simultaneously monitor the expression of many genes on nylon membrane is described and characterized. To quantify the expression of genes and to isolate differentially expressed genes, the southern hybridization process on filter membranes was employed. The levels of gene expression were represented by color intensities generated by colorimetric reactions in place of hazardous radioisotopes or costly laser-induced fluorescence detection. The gene expression patterns on nylon membranes were digitized by devices such as an economical flatbed scanner or a digital camera. The quantitative information of gene expression was retrieved by image analysis software. Quantitative comparison of the northern dot-blotting method with the microarray system is described. Applications employing single-color detection as well as dual-color detection to isolate differentially expressed genes among thousands of genes are demonstrated.

Selection of invasive and metastatic subpopulations from a human lung adenocarcinoma cell line.

To better understand the mechanism(s) underlying lung cancer invasion and metastasis, a Transwell invasion chamber was used to select progressively more invasive cancer cell populations from a clonal cell line of human lung adenocarcinoma, CL1. Five sublines with progressive invasiveness, designated CL1-1, CL1-2, CL1-3, CL1-4, and CL1-5, were obtained through this in vitro selection process. Their invasive abilities through basement membrane matrix showed a 4- to 6-fold increase over that of the parental cells. Moreover, the sublines manifested an increase in their colony-forming ability on soft agar, tumorigenicity, and metastatic potency in severe combined immunodeficiency (SCID) mice. Examining the phenotypes of the cell lines revealed increased expression of 92 kD gelatinase and an increase in the cell population stained with anti-keratin-8 and -18 antibodies. Clonal isolation of anti-keratin-18-antibody-positive and -negative cell populations demonstrated a correlated enhancement of the invasiveness of these cells and their expression of keratin-18. These results support the notion that the metastatic behavior of lung cancer cells can be characterized with this in vitro system, and that the properties of these progressively invasive cancer cells can be clonally studied.

Extracellular pH affects regulation of the pcbAB gene in Penicillium chrysogenum.

The effect of extracellular pH and dissolved oxygen on regulation of the pcbAB gene in P. chrysogenum was examined, using Northern analysis and a reporter gene fusion. It was found that ambient pH markedly affected levels of pcbAB mRNA whereas maintenance of dissolved oxygen concentration above 10% had no detectable effect. The presence of a DNA-binding protein, which binds upstream of the pcbAB translational start codon, was also related to ambient pH. In all fermentations, pcbAB mRNA was most abundant at around the late exponential/early stationary phase of a culture.

Role of intermediate filaments in migration, invasion and metastasis.

The expression of intermediate filament proteins is remarkably tissue-specific which suggests that the intermediate filament (IF) type(s) present in cells is somehow related to their biological function. However, in some cancers-particularly malignant melanoma and breast carcinoma, there is a strong indication that vimentin and keratin IFs are coexpressed, thus presenting as a dedifferentiated or interconverted (between epithelial and mesenchymal) phenotype. In this review, two in vitro models are presented which recapitulate the interconverted phenotype in human melanoma and breast carcinoma, and allow, for the first time, unique observations to be made with respect to the role of IFs in cancer progression. These studies have provided direct evidence linking overexpression of keratin IFs in human melanoma with increased migratory and invasive activity in vitro, which can be down-regulated by substituting dominant-negative keratin mutants. Overexpression of vimentin IFs in the breast carcinoma model leads to augmentation of motility and invasiveness in vitro, which can be transiently down-regulated by treatment with antisense oligonucleotides to vimentin. Additional experimental evidence suggests that the mechanism(s) responsible for the differential expression of metastatic properties associated with the interconverted phenotype rest(s) in the unique interaction, either direct or indirect, of IFs with specific integrins interacting with the extracellular matrix. In this review, we discuss the observations derived from the human melanoma and breast carcinoma models to address the hypothesis that the ability to coexpress vimentin and keratins confers a selective advantage to tumor cells in their interpretation of and response to signaling cues from the extracellular matrix. The ramifications of these observations are discussed with respect to the patholophysiology of the respective in situ tumors.

Acidic pH enhances the invasive behavior of human melanoma cells.

As a consequence of poor perfusion and elevated acid production, the extracellular pH (pHex) of tumors is generally acidic. Despite this, most in vitro experiments are still performed at the relatively alkaline pHex of 7.4. This is significant, because slight changes in pHex can have profound effects on cell phenotype. In this study we examined the effects of mildly acidic conditions on the in vitro invasive potential of two human melanoma cell lines; the highly invasive C8161, and poorly invasive A375P. We observed that culturing of either cell line at acidic pH (6.8) caused dramatic increases in both migration and invasion, as measured with the Membrane Invasion Culture System (MICS). This was not due to a direct effect of pH on the invasive machinery, since cells cultured at normal pH (7.4) and tested at acidic pH did not exhibit increased invasive potential. Similarly, cells cultured at acidic pH were more aggressive than control cells when tested at the same medium pH. These data indicate that culturing of cells at mildly acidic pH induces them to become more invasive. Since acid pH will affect the intracellular pH (pHin) and intracellular calcium ([Ca2+]in), we examined the effect of these parameters on invasion. While changes in [Ca2+]in were not consistent with invasive potential, the changes in pHin were. While these conditions decrease the overall amount of gelatinases A and B secreted by these cells, there is a consistent and significant increase in the proportion of the activated form of gelatinase B.

Experimental coexpression of vimentin and keratin intermediate filaments in human melanoma cells augments motility.

Intermediate filaments have been used as cell-type-specific markers in differentiation and pathology; however, recent reports have demonstrated the coexpression of vimentin (a mesenchymal marker) and keratins (epithelial markers) in numerous neoplasms, including melanoma, which has been linked to metastatic disease. To test the hypothesis that coexpression of vimentin and keratins by melanoma cells contributes to a more migratory and invasive phenotype, we co-transfected a vimentin-positive human melanoma cell line, A375P (of low invasive ability), with cDNAs for keratins 8 and 18. The resultant stable transfectants expressed vimentin- and keratin-positive intermediate filaments showed a two- to threefold increase in their invasion of basement membrane matrix and migration through gelatin in vitro. These findings were further corroborated by video cinematography. During attachment and spreading on fibronectin, the transfectants containing vimentin and keratins 8 and 18 demonstrated an increase in focal adhesions that stained positive for beta 1 integrin and phosphotyrosine, along with enhanced membrane ruffling and actin stress fiber formation. From these data, we postulate that coexpression of vimentin and keratins results in increased cytoskeletal interactions at focal contacts within extracellular matrices involving integrin cell signaling events, which contributes to a more migratory behavior.

Detection of a protein which binds specifically to the upstream region of the pcbAB gene in Penicillium chrysogenum.

The upstream region of the pcbAB gene from Penicillium chrysogenum was screened for protein-binding sites using an electromobility shift assay. A specific protein/DNA interaction was detected within a fragment covering the region -387 to -242 relative to the pcbAB translational start codon. The appearance of this protein and pcbAB mRNA in culture extracts occurred at the same time point in fermentations, suggesting that the protein might be a transcription activator. The putative upstream activating sequence was located more precisely using cross-competition assays. These indicated the involvement of the 7-bp motif TGCCAAG in the binding of the protein.

Somatic mutation of human immunoglobulin V genes in the X-linked HyperIgM syndrome.

Somatic mutation of Ig variable regions occurs prominently in germinal centers, but it has been debated whether the mutation process initiates in germinal centers or is activated before germinal center entry of B cells. We have analyzed for the presence of somatic mutation in Ig gene rearrangements of the nonpolymorphic human VH6 gene in the X-linked HyperIgM syndrome, which is associated with defective CD40 ligand expression and absence of germinal centers and generation of memory B lymphocytes. IgM and rare IgG VH6 productive rearrangements were isolated from PBL of patients with X-linked HyperIgM syndrome. Although the majority of both the IgM and IgG VH6 rearrangements had a germline VH6 sequence, 7 of 102 VH6 IgM and 1 of 6 IgG rearrangements had a mutated VH6 gene. The mutation frequency (mutations/bp) was 1.4% with a range of 2-9 mutations per clone, a mutation frequency lower, however, than that observed in IgM (3.2%) and IgG (5.4%) VH6 rearrangements of normal individuals. These results suggest that somatic mutation may be initiated in a CD40 ligand-independent pathway before entry of B cells into germinal centers, but fails to achieve the high mutation frequency observed in the presence of germinal centers.