HSP90 Inhibition and Cellular Stress Elicits Phenotypic Plasticity in Hematopoietic Differentiation.

Cancer cells exist in a state of Darwinian selection using mechanisms that produce changes in gene expression through genetic and epigenetic alteration to facilitate their survival. Cellular plasticity, or the ability to alter cellular phenotype, can assist in survival of premalignant cells as they progress to full malignancy by providing another mechanism of adaptation. The connection between cellular stress and the progression of cancer has been established, although the details of the mechanisms have yet to be fully elucidated. The molecular chaperone HSP90 is often upregulated in cancers as they progress, presumably to allow cancer cells to deal with misfolded proteins and cellular stress associated with transformation. The objective of this work is to test the hypothesis that inhibition of HSP90 results in increased cell plasticity in mammalian systems that can confer a greater adaptability to selective pressures. The approach used is a murine in vitro model system of hematopoietic differentiation that utilizes a murine hematopoietic stem cell line, erythroid myeloid lymphoid (EML) clone 1, during their maturation from stem cells to granulocytic progenitors. During the differentiation protocol, 80%-90% of the cells die when placed in medium where the major growth factor is granulocyte-macrophage-colony stimulating factor. Using this selection point model, EML cells exhibit increases in cellular plasticity when they are better able to adapt to this medium and survive. Increases in cellular plasticity were found to occur upon exposure to geldanamycin to inhibit HSP90, when subjected to various forms of cellular stress, or inhibition of histone acetylation. Furthermore, we provide evidence that the cellular plasticity associated with inhibition of HSP90 in this model involves epigenetic mechanisms and is dependent upon high levels of stem cell factor signaling. This work provides evidence for a role of HSP90 and cellular stress in inducing phenotypic plasticity in mammalian systems that has new implications for cellular stress in progression and evolution of cancer.

17-N-Allylamino-17-demethoxygeldanamycin induces a diverse response in human acute myelogenous cells.

The goal of this study was to ascertain the specific effects of 17-N-Allylamino-17-demethoxygeldanamycin (17-AAG) treatment in human acute myelogenous leukemia (AML). Four human leukemia cell lines were treated with varying doses of 17-AAG followed by analysis of toxicity, apoptosis, proliferation, and cell cycle. Cell cycle analysis revealed that the cells accumulate in G2/M phase within 96 h of treatment, although the effect was not equivalent among the cell lines. p21, p53 expression and MDR1 activity were among the possible mechanisms uncovered for the differing responses. Exploiting these differences may allow for more effective combinatory treatments in patients with AML.

Lipotoxin F of Pseudomonas aeruginosa is an AlgU-dependent and alginate-independent outer membrane protein involved in resistance to oxidative stress and adhesion to A549 human lung epithelia.

Chronic lung infection with P. aeruginosa and excessive neutrophil-associated inflammation are major causes of morbidity and mortality in patients with cystic fibrosis (CF). Overproduction of an exopolysaccharide known as alginate leads to the formation of mucoid biofilms that are resistant to antibiotics and host defences. Alginate overproduction or mucoidy is controlled by a stress-related ECF sigma factor AlgU/T. Mutation in the anti-sigma factor MucA is a known mechanism for conversion to mucoidy. Recently, we showed that inactivation of a kinase (KinB) in nonmucoid strain PAO1 results in overproduction of alginate. Here, we report the initial characterization of lipotoxin F (LptF, PA3692), an OmpA-like outer membrane protein that exhibited increased expression in the mucoid PAO1kinB mutant. The lipotoxin family of proteins has been previously shown to induce inflammation in lung epithelia, which may play a role in CF disease progression. Expression of LptF was observed to be AlgU-dependent and upregulated in CF isolates. Deletion of lptF from the kinB mutant had no effect on alginate production. Deletion of lptF from PAO1 caused a differential susceptibility to oxidants that can be generated by phagocytes. The lptF and algU mutants were more sensitive to hypochlorite than PAO1. However, the lptF mutant displayed increased resistance to hydrogen peroxide. LptF also contributed to adhesion to A549 human lung epithelial cells. Our data suggest that LptF is an outer membrane protein that may be important for P. aeruginosa survival in harsh environments, including lung colonization in CF.

Defect in early lung defence against Pseudomonas aeruginosa in DBA/2 mice is associated with acute inflammatory lung injury and reduced bactericidal activity in naive macrophages.

Pseudomonas aeruginosa is an opportunistic pathogen that causes serious respiratory disease in the immune-compromised host. Using an aerosol infection model, 11 inbred mouse strains (129/Sv, A/J, BALB/c, C3H/HeN, C57BL/6, DBA/2, FVB, B10.D2/oSnJ, B10.D2/nSnJ, AKR/J and SWR/J) were tested for increased susceptibility to P. aeruginosa lung colonization. DBA/2 was the only mouse strain that had increased bacterial counts in the lung within 6 h post-infection. This deficiency incited a marked inflammatory response with reduced bacterial lung clearance and a mortality rate of 96.7 %. DBA/2 mice displayed progressive deterioration of lung pathology with extensive alveolar exudate and oedema formation at 48-72 h post-infection. The neutrophil-specific myeloperoxidase activity remained elevated throughout infection, suggesting that the increased leukocyte infiltration into alveoli caused acute inflammatory lung injury. DBA/2 mice lack the haemolytic complement; however, three additional mouse strains (AKR/J, SWR/J and A/J) with the same defect effectively cleared the infection, indicating that other host factors are involved in defence. Bone marrow-derived macrophages of DBA/2 showed an initial increase in phagocytosis, while their bactericidal activity was reduced compared to that of C57BL/6 macrophages. Comparison of pulmonary cytokine profiles of DBA/2 versus C57BL/6 or C3H/HeN indicated that DBA/2 had similar increases in tumour necrosis factor (TNF)-alpha, KC and interleukin (IL)-1a as C3H/HeN, but showed specific induction of IL-17, monocyte chemotactic protein (MCP)-1 and vascular endothelial growth factor (VEGF). Together, DBA/2 mice have a defect in the initial lung defence against P. aeruginosa colonization, which causes the host to produce a greater, but damaging, inflammatory response. Such a response may originate from the reduced antimicrobial activity of DBA/2 macrophages.