IGFBP7 induces apoptosis of acute myeloid leukemia cells and synergizes with chemotherapy in suppression of leukemia cell survival

Despite high remission rates after chemotherapy, only 30–40% of acute myeloid leukemia (AML) patients survive 5 years after diagnosis. This extremely poor prognosis of AML is mainly caused by treatment failure due to chemotherapy resistance. Chemotherapy resistance can be caused by various features including activation of alternative signaling pathways, evasion of cell death or activation of receptor tyrosine kinases such as the insulin growth factor-1 receptor (IGF-1R). Here we have studied the role of the insulin-like growth factor-binding protein-7 (IGFBP7), a tumor suppressor and part of the IGF-1R axis, in AML. We report that IGFBP7 sensitizes AML cells to chemotherapy-induced cell death. Moreover, overexpression of IGFBP7 as well as addition of recombinant human IGFBP7 is able to reduce the survival of AML cells by the induction of a G2 cell cycle arrest and apoptosis. This effect is mainly independent from IGF-1R activation, activated Akt and activated Erk. Importantly, AML patients with high IGFBP7 expression have a better outcome than patients with low IGFBP7 expression, indicating a positive role for IGFBP7 in treatment and outcome of AML. Together, this suggests that the combination of IGFBP7 and chemotherapy might potentially overcome conventional AML drug resistance and thus might improve AML patient survival.Only 30–40% of acute myeloid leukemia (AML) patients survive 5 years after diagnosis.¹ This extremely poor prognosis is mainly caused by treatment failure due to chemotherapy resistance. This resistance is often a multifactorial phenomenon that can include enhanced expression or activation of receptor tyrosine kinases such as the insulin growth factor-1 receptor (IGF-1R).^{2, 3} The IGF-1R stimulates proliferation, protects cells from apoptosis and has been implicated in the development and maintenance of various cancers.^{4, 5} Several oncogenes require an intact IGF-1R pathway for their transforming activity⁶ and moreover, disruption or inhibition of IGF-1R activity has been shown to inhibit the growth and motility of a wide range of cancer cells in vitro and in mouse models.^{4, 5} IGF-1Rs are membrane receptors and binding of their ligand, the insulin-like growth factor-1 (IGF-1), results in receptor phosphorylation and activation of MAPK and PI3K/Akt signaling.⁴ Importantly, IGF-1, normally produced by the liver and bone marrow stromal cells, can stimulate the proliferation of cancer cells in vitro and genetic manipulations that reduce IGF-1 signaling can lead to decreased tumor growth.^{7, 8}In hematological malignancies, a role for IGF-1 signaling has been demonstrated in multiple myeloma (MM) where it stimulates growth and potently mediates survival.⁹ Several anti-IGF-1R strategies have been shown to inhibit MM growth.^{10, 11} In AML, expression of the IGF-1R and IGF-1 was detected in AML cell lines and primary AML blasts and stimulation with IGF-1 can promote the growth of AML cells.^{12, 13, 14} In addition, neutralizing IGF-1R antibodies and the tyrosine kinase inhibitors (TKIs) NVP-AEW541 and NVP-ADW742, have been shown to inhibit proliferation and to induce apoptosis.^{15, 16}In addition to its mitogenic and anti-apoptotic roles, directly influencing tumor development, IGF-1R appears to be a critical determinant of response to numerous anti-cancer therapies, including TKIs and chemotherapy.^{2, 3, 17, 18, 19, 20, 21, 22} In AML, activated IGF-1R signaling has been linked to cytarabine resistance, a drug included in every AML treatment schedule.¹⁷ Notably, in several cancer cell lines, a small subpopulation of drug-tolerant cancer cells exists that maintains their viability, after treatment with a lethal drug dose, via engagement of the IGF-1R.¹⁸The activity of the IGF-1R is tightly controlled at multiple levels, including their processing, endocytosis, trafficking and availability of its ligands.⁴ Ligand bioavailability is partly controlled by the family of secreted insulin-like growth factor-binding protein (IGFBP1 to IGFBP6), which can bind to IGFs therewith regulating the interaction of these ligands to their receptors. However, as IGFBPs are able to induce IGF-dependent and IGF-independent effects, the results of several studies on their role in cancer cell survival appeared to be controversial and complex.^{23, 24} In addition to IGFBPs, various IGFBP-related proteins have been identified.^{23, 25} One of these is the IGFB-related protein 1, also known as insulin-like growth factor-binding protein-7 (IGFBP7). IGFBP7 has 30% homology to IGFBP1 to IGFBP6 in its N-terminal domain and functions predominantly as a tumor suppressor.^{23, 24, 25, 26} In contrast to IGFBP1 to IGFBP6, which bind to the IGFs,²³ IGFBP7 is a secreted protein that can directly bind to the IGF-1R and thereby inhibits its activity.²⁷ The abundance of IGFBP7 is inversely correlated with tumor progression in hepatocellular carcinoma.²⁸ Importantly, decreased expression of IGFBP7 has been associated with therapy resistance^{29, 30} and increasing IGFBP7 levels can inhibit melanoma and breast cancer growth.^{31, 32} IGFBP7 was originally identified as being involved in Raf-mediated apoptosis and senescence³³ and also has been shown to induce senescence in mesenchymal stromal cells.³⁴We established that IGFBP7 induces a cell cycle block and apoptosis in AML cells and cooperates with chemotherapy in the induction of leukemia cell death. AML patients with low IGFBP7 expression have a worse outcome than patients with high IGFBP7 expression, indicating that AML patients might benefit from a combination therapy consisting of chemotherapy and IGFBP7. Our results define IGFBP7 as a focus to enhance chemotherapy efficacy and improve AML patient survival.     

Association of genetic variants rs641153 (CFB), rs2230199 (C3), and rs1410996 (CFH) with age-related macular degeneration in a Brazilian population

This study aimed to investigate the association among genetic variants of the complement pathway CFB R32Q (rs641153), C3 R102G (rs2230199), and CFH (rs1410996) with age-related macular degeneration (AMD) in a sample of the Brazilian population. In a case-control study, 484 AMD patients were classified according to the clinical age-related maculopathy grading system (CARMS) and compared to 479 unrelated controls. The genetic variants rs1410996 of complement H (CFH), rs641153 of complement factor B (CFB), and rs2230199 of complement 3 (C3) were evaluated through polymerase chain reaction (PCR) and direct sequencing. The associations between single nucleotide polymorphisms (SNPs) and AMD, adjusted by age, were assessed by using logistic regression models. A statistically significant association was observed between AMD risk and rs2230199 variant with an OR of 2.01 (P  = 0.0002) for CG individuals compared to CC individuals. Regarding the comparison of advanced AMD versus the control group, the OR was 2.12 (P = 0.0036) for GG versus AA genotypes for rs1410996 variant. Similarly, the OR for rs2230199 polymorphism was 2.3034 (P  = 5.47^e-05) when comparing CG individuals to CC carriers. In contrast, the rs641153 variant showed a significant protective effect against advanced AMD for GA versus GG genotype (OR = 0.4406; P  = 0.0019). When comparing wet AMD versus controls, a significant association was detected for rs1410996 variant (OR = 2.16; P  = 0.0039) comparing carriers of the homozygous GG versus AA genotype, as well as in the comparisons of GG (OR = 3.0713; P  = 0.0046) and CG genotypes (OR = 2.2249; P  = 0.0002) versus CC genotype for rs2230199 variant, respectively. The rs641153 variant granted a significant protective effect against wet AMD for GA versus GG genotypes (OR = 0.4601; P  = 0.0044). Our study confirmed the risk association between rs2230199 and rs1410996 variants and AMD, and the protective role against AMD for rs641153 variant.     

Development of multiple strain competitive index assays for <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> using pIMC; a new site-specific integrative vector

Background  

The foodborne, gram-positive pathogen, Listeria monocytogenes, is capable of causing lethal infections in compromised individuals. In the post genomic era of L. monocytogenes research, techniques are required to identify and validate genes involved in the pathogenicity and environmental biology of the organism. The aim here was to develop a widely applicable method to tag L. monocytogenes strains, with a particular emphasis on the development of multiple strain competitive index assays.     

Use of spectral analysis to test hypotheses on the origin of pinnipeds

The evolutionary origin of the pinnipeds (seals, sea lions, and walruses) is still uncertain. Most authors support a hypothesis of a monophyletic origin of the pinnipeds from a caniform carnivore. A minority view suggests a diphyletic origin with true seals being related to the mustelids (otters and ferrets). The phylogenetic relationships of the walrus to other pinniped and carnivore families are also still particularly problematic. Here we examined the relative support for mono- and diphyletic hypotheses using DNA sequence data from the mitochondrial small subunit (12S) rRNA and cytochrome b genes. We first analyzed a small group of taxa representing the three pinniped families (Phocidae, Otariidae, and Odobenidae) and caniform carnivore families thought to be related to them. We inferred phylogenetic reconstructions from DNA sequence data using standard parsimony and neighbor-joining algorithms for phylogenetic inference as well as a new method called spectral analysis (Hendy and Penny) in which phylogenetic information is displayed independently of any selected tree. We identified and compensated for potential sources of error known to lead to selection of incorrect phylogenetic trees. These include sampling error, unequal evolutionary rates on lineages, unequal nucleotide composition among lineages, unequal rates of change at different sites, and inappropriate tree selection criteria. To correct for these errors, we performed additional transformations of the observed substitution patterns in the sequence data, applied more stringent structural constraints to the analyses, and included several additional taxa to help resolve long, unbranched lineages in the tree. We find that there is strong support for a monophyletic origin of the pinnipeds from within the caniform carnivores, close to the bear/raccoon/panda radiation. Evidence for a diphyletic origin was very weak and can be partially attributed to unequal nucleotide compositions among the taxa analyzed. Subsequently, there is slightly more evidence for grouping the walrus with the eared seals versus the true seals. A more conservative interpretation, however, is that the walrus is an early, but not the first, independent divergence from the common pinniped ancestor.      

Impact of Nutrient Composition on a Degradative Biofilm Community

A microbial community was cultivated in flow cells with 2,4,6-trichlorobenzoic acid (2,4,6-TCB) as sole carbon and energy source and was examined with scanning confocal laser microscopy and fluorescent molecular probes. The biofilm community which developed under these conditions exhibited a characteristic architecture, including a basal cell layer and conspicuous mounds of bacterial cells and polymer (approximately 20 to 30 (mu)m high and 25 to 40 (mu)m in diameter) occurring at 20- to 200-(mu)m intervals. When biofilms grown on 2,4,6-TCB were shifted to a labile, nonchlorinated carbon source (Trypticase soy broth), the biofilms underwent an architectural change which included the loss of mound structures and the formation of a more homogeneous biofilm. Neutrally charged fluorescent dextrans, which upon hydration become cationic, were observed to bind to mounds, as well as to the basal cell layer, in 14-day biofilms. In contrast, polyanionic dextrans bound only to the basal cell layer, indicating that this material incorporated sites with both positive and negative charge. The results from this study indicate that nutrient composition has a significant impact on both the architecture and the physicochemistry of degradative biofilm communities.     

Multicellular Organization in a Degradative Biofilm Community

Diclofop methyl, a commercial herbicide, was used as the sole carbon source to cultivate diclofop-degrading biofilms in continuous-flow slide culture. The biofilms were analyzed by using scanning confocal laser microscopy and image analysis. Spatial relationships among members of the community were distinctive to diclofop-grown biofilms. These relationships did not develop when the biofilms were grown on more labile substrates but were conserved when the biofilms were cultivated with other chlorinated ring compounds. The structures included conical bacterial consortia rising to 30 μm above the surrounding biofilm, grape-like clusters of cocci embedded in a matrix of perpendicularly oriented bacilli, and other highly specific patterns of intra- and intergeneric cellular coaggregation and growth. These unique consortial relationships indicated that syntrophic interactions may be necessary for optimal degradation of diclofop methyl and other chlorinated ring compounds.     

Assessment of the working range and effect of sodium dichloroisocyanurate on Pseudomonas aeruginosa biofilms and planktonic cells

Sodium dichloroisocyanurate (NaDCC) is a chemical agent that acts against microorganisms in a manner similar to that of sodium hypochlorite by releasing free available chlorine. NaDCC has been approved by the WHO for the emergency treatment of water and by the US EPA for routine treatment of water. Previous studies assessing the effectiveness of NaDCC for the treatment of water implied that NaDCC should have a wide array of disinfecting effects beyond the treatment of planktonic cells in potable water. In this study the biocidal effects of NaDCC against Pseudomonas aeruginosa cells in different growth modes including planktonic cells and biofilms were explored. The data showed that a 60% dilution of the standard NaDCC solution was effective in the treatment of both P. aeruginosa planktonic cells and biofilms.     

Metabolic Differentiation in Biofilms as Indicated by Carbon Dioxide Production Rates

The measurement of carbon dioxide production rates as an indication of metabolic activity was applied to study biofilm development and response of Pseudomonas sp. biofilms to an environmental disturbance in the form of a moving air-liquid interface (i.e., shear). A differential response in biofilm cohesiveness was observed after bubble perturbation, and the biofilm layers were operationally defined as either shear-susceptible or non-shear-susceptible. Confocal laser scanning microscopy and image analysis showed a significant reduction in biofilm thickness and biomass after the removal of the shear-susceptible biofilm layer, as well as notable changes in the roughness coefficient and surface-to-biovolume ratio. These changes were accompanied by a 72% reduction of whole-biofilm CO₂ production; however, the non-shear-susceptible region of the biofilm responded rapidly after the removal of the overlying cells and extracellular polymeric substances (EPS) along with the associated changes in nutrient and O₂ flux, with CO₂ production rates returning to preperturbation levels within 24 h. The adaptable nature and the ability of bacteria to respond to environmental conditions were further demonstrated by the outer shear-susceptible region of the biofilm; the average CO₂ production rate of cells from this region increased within 0.25 h from 9.45 ± 5.40 fmol of CO₂·cell⁻¹·h⁻¹ to 22.6 ± 7.58 fmol of CO₂·cell⁻¹·h⁻¹ when cells were removed from the biofilm and maintained in suspension without an additional nutrient supply. These results also demonstrate the need for sufficient monitoring of biofilm recovery at the solid substratum if mechanical methods are used for biofouling control.Spatial differences in biofilm cohesiveness have been observed after the application of increased shear forces. Coufort et al. (8) subjected both aerobic and anaerobic biofilms, cultivated on ethanol or wastewater, to increased shear stress and found that the biofilm layer at the bulk liquid interface was removed by slight increases in shear rates (0.2 Pa), whereas the middle and base biofilm layers were able to resist removal when exposed to up to 2 Pa and 13 Pa, respectively (8). Total organic carbon (TOC) analyses indicated that the sensitive top layer of the biofilm contained approximately 60% of the total biofilm biomass while the remaining two layers each represented approximately 20%. In a follow-up study, biofilms grown under similar conditions exhibited comparable degrees of heterogeneity in the susceptibility of the various biofilm layers to shear and abrasion (9). It was also indicated that the basal biofilm layer contained active microorganisms, as characterized by oxygen uptake rates, but no details were provided on the methodology or time lapse after the removal of the less-cohesive upper biofilm layers.Spatial differentiation in metabolic activity in biofilms has also been noted. Most experimental strategies to determine biofilm activity have been centered on microscopy in combination with fluorescent reporter genes or probes that target various indicators of physiological activity in the cell. Several fluorescent stains have been applied previously, such as 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) (15) and acridine orange (27) as well as the commercially available BacLight viability kit (17). Reporter gene expression is another means to evaluate physiological activity in a biofilm. Alkaline phosphatase activity correlated well with oxygen penetration into the upper layers (30 μm) of 117- to 151-μm-thick biofilms (28).Although all of the above approaches have been shown to be effective, most suffer from inherent disadvantages (26), including incomplete penetration of fluorescent stains and the production of artifacts, and, perhaps most significantly, generally allow only end point analysis due to cellular toxicity. Reporter gene technologies may circumvent this problem but require prior genetic manipulation, and it is unknown what, if any, changes in cell physiology may occur as a result of expression of the reporter gene. The need for genetic manipulation further constrains analysis to pure culture studies.The basis for spatial heterogeneity in biofilm physiological activity is widely accepted, as previously reviewed (25, 26). Limited diffusion of nutrients and oxygen into the biofilm from the bulk liquid and waste products from a multilayered biofilm are among the simplest explanations since the absence of a complete exchange with the environment, in concert with microbial activity, leads to the formation of chemical gradients in the biofilm. The bacteria in the biofilm respond to the gradients, likely by altering gene expression patterns as determined by global regulators. The remarkable recalcitrance of biofilms toward many antimicrobials may in part be due to the insensitivity of dormant cells in the regions of the biofilm where limited diffusion reduces metabolic activity.The effect of air bubbles on biofilm stability has mostly been studied in a dental context, where biofilm removal is the goal. Gomez-Suarez et al. (11) utilized a single bubble to investigate the strength of bacterial cell adhesion to various surfaces (11). According to the authors, the probability of cell detachment due to the movement of an air bubble over an attached cell is determined by several factors, namely, collision efficiency, bubble-bacteria attachment efficiency, and the stability of the bubble-bacteria aggregate. For a bubble spanning the entire width of a flow chamber, the collision efficiency is expected to be equal to 1 although the velocity of the bubble may also influence the detachment efficiency since a rapidly moving bubble will result in a thicker liquid film surrounding the bubble, which in turn decreases the collision efficiency. Bacterium-substratum adhesion forces of ∼10⁻⁹ N were estimated, which is significantly smaller than the detachment force of a bubble moving over an attached cell (up to 10⁻⁷ N).Liquid flow in most environments—in nature, industry, and clinical or dental settings—typically shows much variation. It can be expected that microbial biofilms have evolved to manage this variability and even to utilize the resulting differences in flow to optimize activity (e.g., the prevention of excessive biomass accumulation for the maintenance of optimum gradients of nutrients and gases) or to relocate to more favorable environments.Furthermore, increased shear is a recognized strategy to remove unwanted microbial growth from surfaces; therefore, methods to measure the effect of shear on biofilms, including biofilm recovery after partial shear-induced removal, should contribute to our overall understanding of this important form of microbial existence. We developed an approach that measures CO₂ production as an indication of biofilm activity in real time and combined this method with confocal laser scanning microscopy (CLSM) and cell yield measurements to study activity-structure relationships in biofilms. This approach is an extension of the one we described in 2009 (18) and enables us to comment on differences in metabolic activity of the whole biofilm versus that of the shear-susceptible biofilm region and to compare biofilm-derived planktonic cells with those growing in batch culture.